vorgelegt von - connecting repositories · 2016-06-11 · alam, saeed gul, imran, hafeez, waqas,...
TRANSCRIPT
Induction of Recombinant Proteins in Wheat Seed and Vegetative Tissues
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen
Grades eines Doktors der Naturwissenschaften
genehmigte Dissertation
vorgelegt von
Mag. Phil. in Biotechnologie und Genetechnik
Imran Khan
aus Peshawar, Pakistan
Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer
Universitätsprofessor Dr. rer. nat. Eva Stoger
Tag der mündlichen Prüfung: 20.02.2013
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar
Dedication
I feel it a great honour to dedicate my PhD thesis to my
beloved parents for their significant contribution in
achieving this goal of academic excellence.
Acknowledgements
I am, first of all, extremely thankful to Almighty Allah whose blessing have always
followed me regardless of the fact weather I deserved or not. It always seems to me a
miracle of very convoluted nature to see ordinary people like me, doing unordinary things.
I express my deepest gratitude for whatever I have been bestowed with by the gracious.
I am in grateful to Prof. Dr. Rainer Fischer for supervising my PhD and to provide me
every possible help and opportunity to work at Institute of Molecular Biotechnology,
RWTH Aachen. I am thankful to him for giving me opportunity to join his group and
commence research in the area that captured my interest and imagination at first glance.
I‟m greatly honoured to pay my deep gratitude to Prof. Dr. Eva Stöger under whose
stimulating guidance, valuable suggestions, sympathetic attitude and encouragements this
research work was carried out. Eva is someone you will instantly love and never forget
once you meet her. Although I was supposed to stay at RWTH Aachen for the entire
duration of my PhD but when Eva moved to Vienna, I couldn‟t resist the temptation to
move there as well despite all the troubles of moving from one country to another. I wish
that I could be as lively, kind, enthusiastic, and energetic as Eva.
I am indebted to Dr. Elsa Arcalis for her discussions, support, editorial judgement and
insightful contribution to lend finesse to every aspect of my draft. Dear Elsa, thank you so
much, with your constant care and motivation, I was able to finish this PhD which
otherwise would have taken I don‟t know how many years. When I was writing my thesis,
your input was of the greatest value and I am so thankful to you for helping out in the labs
and experiments. Fewer people have I seen having a friendlier nature than you have got.
God bless you with a very happy life ahead.
How can I forget to mention Dr. Thomas Rademacher and Markus Sack for being the
person who fed me with the knowledge and skills required to do the experimental and
empirical work in the labs in Aachen. Sincerest gratitude to for their dynamic and
purposeful support, literally skill, distinguished and enthusiastic guidance in a very
friendly environment provided me a confidence to pursuit this valuable research work.
It wouldn‟t be fair to miss out on appreciating certain other people‟s help. Uli and Verena
provided not only help but an amazing company during the time I was working in Vienna.
Certain colleagues became some of my deepest friends. I convey my heartiest and
sincerest acknowledgements to Helene, Stanislov, Jenny, Tuba, Sneha, Hue, Gabi, Julian,
Valerie, Ester and Anna were the people whose presence around me was so monumental
both for my research as well as my social life. I can‟t forget the amazing time we had. Life
was so colourful because of your presence. You guys are the coolest friend one can ever
find! Thanks for bearing with me when I called you „Hell‟ dear Helene and “everybody‟s
LOVE” dear Stanislov.
Donatella, David, Kristina, Eva, Kasia are the ever best friends at Vienna Institute for
Biotechnology. Thank you so much for providing me an amazing social circle to enjoy my
free time. I never loved going to the kitchen for cooking but it was your company that
made me staying longer there and eventually a good cook too.
During my stay at Aachen, Germany, some of my compatriots in Aachen were a great
company. Noor, Aamer, Wasif, Mubasher, Usman, Falak, Adnan, Abid, Arham and
Rizwan all of you were amazing. I never thought I would meet the coolest countrymen in
a foreign country. Thank you all for being such a great family.
Some friends from Bremen, Geremany were great whenever I visited them. Abu Nasar,
Asim, Sadiq, Wakeel, Nawab, Noor Shad and Amna. I will miss the great time we spent
together.
Some people in Vienna, Austria that I wouldn‟t miss out on mentioning here are Fakh-e-
alam, Saeed gul, Imran, Hafeez, Waqas, Rehan, Hammad, Tahir, GM, Usman, Hameed,
Shaghif, Elazibath, Ana, Eva and Gosia. What a memorable time we spent together. Thank
you all for your love and time.
Finally but immensely, I owe major debts to “The backbone of my life” my loving parents
and caring brothers and sisters, whose immeasurable support and eminent understanding
has helped me through thick and thin. With their inexplicable contributions only, I have
reached this stage. My father, who had this dream that his son becomes a PhD doctor, will
be so proud seeing this effort. It was indeed his love and the hard work he always did in
his life so to give us a better living and a future that I have today. My mother, whose love
is unmatchable and whose heart beats but with mine always. Thank you so much to m
brothers, who never let me do the chores so that I could focus on my studies, and to my
sisters who are the most loving and adorable sisters in the world.
Finally, I wish to thank Higher Education Commission (HEC), Islamabad Pakistan for
their financial support and Germany Academic Exchange Service (DAAD) Germany for
organizational help in the entire duration of doctoral studies. Thanks to Institute for
Molecular Biotechnology, RWTH Aachen, Germany and Vienna Institute for
Biotechnology (VIBT), BOKU Vienna, Austria for hosting me.
Imran Khan
29th
October, 2012
Aachen, Germany
i
Contents
Dedication ............................................................................................................ ii
List of Figures ................................................................................................... iv
List of Tables ........................................................................................................ v
Abstract .............................................................................................................. vi
I. Introduction .............................................................................................. 1
I.1. Molecular farming .................................................................................................. 1
I.2. The seed .................................................................................................................. 2
I.2.1. Seed storage proteins ......................................................................................... 3
I.3. Seed as bioreactor ................................................................................................... 4
I.3.1. Wheat ................................................................................................................. 5
I.4. Subcellular protein targeting................................................................................... 5
I.4.1. Endoplasmic reticulum ...................................................................................... 6
I.4.2. Protein bodies and protein storage vacuoles ...................................................... 8
I.4.3. Oil bodies ........................................................................................................... 8
I.4.4. Starch binding domains...................................................................................... 9
I.5. Seed storage protein trafficking ............................................................................ 10
I.6. Fluorescent protein markers ................................................................................. 14
I.6.1. Discosoma red fluorescent protein................................................................... 15
I.7. Encapsulation of recombinant proteins ................................................................ 16
I.7.1. Zein .................................................................................................................. 17
I.7.2. Elastin-like polypeptide ................................................................................... 18
I.7.3. Hydrophobins ................................................................................................... 20
Objectives of the study............................................................................22
II. Materials and methods ........................................................................... 24
II.1. Materials ............................................................................................................... 24
II.1.1. Chemicals and consumables ............................................................................ 24
II.1.2. Buffers, media and solutions............................................................................ 24
II.1.3. Plants ................................................................................................................ 28
II.1.4. Vectors ............................................................................................................. 28
ii
II.1.5. Resin and membranes ...................................................................................... 28
II.1.6. Equipment ........................................................................................................ 28
II.2. Methods ................................................................................................................ 29
II.2.1. Transformation, selection and characterisation of recombinant bacteria ........ 29
II.2.2. Preparation of competent E. coli cells for heat-shock transformation ............. 29
II.2.3. Transformation of E. coli by heat-shock .......................................................... 30
II.2.4. Preparation of competent E. coli cells for electroporation .............................. 30
II.2.5. Transformation of E. coli by electroporation ................................................... 30
II.3. Recombinant DNA techniques ............................................................................. 31
II.3.1. Isolation of plasmid DNA ................................................................................ 31
II.3.2. Restriction enzyme digestion ........................................................................... 31
II.3.3. Agarose gel electrophoresis ............................................................................. 31
II.3.4. Ligation of DNA .............................................................................................. 32
II.3.5. DNA isolation .................................................................................................. 32
II.3.6. Polymerase chain reaction ............................................................................... 33
II.3.7. Purification of DNA from agarose gel ............................................................. 33
II.3.8. Determination of the DNA concentration ........................................................ 34
II.3.9. DNA sequencing .............................................................................................. 34
II.4. Wheat transformation ........................................................................................... 34
II.4.1. Isolation and pre-culture of immature embryo ................................................ 35
II.4.2. Preparation of DNA coated gold particles ....................................................... 35
II.4.3. Micro-projectile bombardment ........................................................................ 36
II.4.4. Tissue culture and selection of transformants .................................................. 36
II.4.5. Selection and propagation of transgenics ........................................................ 40
II.5. Protein analysis ..................................................................................................... 40
II.5.1. Isolation of total soluble proteins from wheat tissues ...................................... 40
II.5.2. SDS-polyacrylamide gel electrophoresis of proteins ....................................... 41
II.5.3. Coomassie brilliant blue staining ..................................................................... 41
II.5.4. Western blot ..................................................................................................... 41
II.6. Immunolocalization studies .................................................................................. 42
II.6.1. Fixation and embedding ................................................................................... 42
II.6.2. Fixation for immunocytochemistry.................................................................. 42
II.6.3. Fixation for ultra-structure ............................................................................... 42
iii
II.6.4. Light and electron microscopy ......................................................................... 44
II.6.5. Immunofluorescent labeling ............................................................................ 44
II.6.6. Fluorescence microscopy ................................................................................. 45
II.6.7. Confocal microscopy ....................................................................................... 45
III. Results ..................................................................................................... 46
III.1. Fusion protein expression construct ..................................................................... 46
III.2. Transformation of wheat plants ............................................................................ 48
III.3. Selection of wheat plants using PPT .................................................................... 51
III.4. Molecular verification of transgenic wheat lines.................................................. 54
III.5. DsRed as a marker for selection and protein trafficking ...................................... 55
III.6. Model fusion protein in wheat tissues .................................................................. 61
III.6.1. Presence and stability of the recombinant protein ........................................... 61
III.6.2. Model fusion protein expression and accumulation in transgenic wheat
leaves................................................................................................................ 63
III.6.3. DsRed mobility in wheat leaf tissues ............................................................... 65
III.6.4. Model fusion protein expression and accumulation in transgenic wheat
endosperm ........................................................................................................ 65
III.6.5. Model fusion protein expression and accumulation in transgenic wheat
embryo ............................................................................................................. 68
IV. Discussion .................................................................................................. 71
V. Conclusion ................................................................................................. 84
VI. References ................................................................................................. 85
Appendix ....................................................................................................... 107
Curriculum Vitae ........................................................................................... 111
iv
List of Figures
Figure I.1 Illustration of the seed anatomy of three plant models. 2
Figure I.2 ER-targeted Green fluorescent protein (GFP). 7
Figure I.3 Wheat A-type and B-type granule structure. 10
Figure I.4 Conceptual diagram of the ontogeny of PBs and PSVs . 13
Figure I.5 Deposition of wheat protein bodies. 13
Figure I.6 Protein bodies deposition in maize and rice. 14
Figure I.7 Red Fluorescent protein as selection marker. 15
Figure I.8 Using storage organelles for the accumulation and encapsulation of recombinant
proteins 17
Figure I.9 Localization of storage proteins in maize endosperm. 18
Figure I.10 Fluorescence microscopic image of Tobacco leaf. 19
Figure I.11 ER-targeted GFP Hydrophobin fusion (GFP-HFBI) promotes the formation of
PBs in Nicotiana benthamiana. 20
Figure III.1 Vector map of pTRAbux-DsRed.zen-H (DsRedzenH). 47
Figure III.2 Structure of the pTRAbux-DsRed-zenH (DsRedzenH) expression vector. 48
Figure III.3 Wheat plant transformation procedure. 49
Figure III.4 Bio-Rad PDS1000/He particle bombardment apparatus. 50
Figure III.5 Wheat tissue culture: from immature embryos to shoot development.. 50
Figure III.6 Wheats plant after PPT application. 52
Figure III.7 Segregation of transgenic wheat lines. 53
Figure III.8 Confirmation of transgene integration by PCR. 54
Figure III.9 Protein pellets extracted from wheat leaves under green light. 56
Figure III.10 DsRed expression during transgenic callus development. 57
Figure III.11. DsRed as a visual selection marker in transgenic wheat lines. 58
Figure III.12 Expression of induced protein bodies in wheat endosperm cells. 59
v
List of Tables
Table I.1 Fusion sequences for the creation of artificial storage organelles. 17
Table I.2 Examples of recombinant proteins produced in cereal seeds 21
Table II.1 Sequence of primers used for DsRed detection. 33
Table II.2 Media ingredients used for wheat tissue culture. 38
Table II.3 List of ingredients and suppliers. 39
Table II.4 MS vitamin ingredients. 39
Table II.5 Series of acetone dehydration for ultra structure fixation. 43
Table II.6 Mixture of resin/ethanol used for ultra structure fixation of the tissues. 43
vi
Abstract
Cereals offer advantages for molecular farming because seeds have naturally evolved for
protein storage. In seeds, the recombinant proteins can be targeted to storage organelles,
such as Protein Bodies (PBs) derived from the endoplasmic reticulum (ER) and Protein
Storage Vacuoles (PSVs) for stable accumulation and storage. γ-Zein, the maize prolamin
storage protein, and partial sequences thereof can be fused to proteins of interest to induce
the formation of novel PB-like organelles not only in its native environment (maize
endosperm) but also in other plant cells and in eukaryotic cells generally. We report the
constitutive expression of recombinant fusion protein i.e. repeated and Pro-X domain of γ-
Zein and red fluorescent protein (DsRed) in transgenic wheat plants. The red fluorescent
protein (DsRed) was used as tool for tracking subcellular localization of recombinant
proteins and also as fluorescent selection marker to monitor transgene expression during
wheat transformation. In the present study we explored endogenous and ectopically
induced protein storage organelles as preferred intracellular destination for recombinant
protein in wheat different tissues i.e. leaf, endosperm and embryo. Our finding has
confirmed that the model fusion protein has induced the formation of an additional
population of protein bodies containing the recombinant protein in seed and vegetative
tissues. To better understand the trafficking of recombinant proteins in ectopic tissues, the
subcellular localization of recombinant proteins in seed and green tissues were examined.
We confirmed the feasibility of using γ-Zein for targeting recombinant protein into
artificial protein bodies those were preferably retained in the protein storage vacuoles
(PSV) in seed tissues. In vegetative tissues, the induced protein bodies were found highly
mobile organelles, exhibiting different pattern of movement with no specific direction.
The presence of the model fusion protein was validated in all three transgenic wheat plant
tissues tested. Also, no degradation products were detected in transgenic wheat seeds after
five months storage. The expression of recombinant fusion proteins had no harmful effect
on the viability and development of transgenic wheat plants.
1
I. INTRODUCTION
I.1. Molecular farming
The concept of molecular farming describes the production of valuable recombinant
proteins at agricultural scale in transgenic plants (Schillberg et al., 2003). In the last two
decades, humans have started exploring different production systems by using plants for
the production of many recombinant proteins. Plants provide an alternative expression
system to microbes or mammalian cells grown in fermentors, which have limited capacity,
higher production costs and in case of bacteria, lack post translational modifications.
Molecular farming has an edge over other production systems for recombinant proteins in
terms of eukaryotic post translational modifications, low risk of human and animal
pathogen contaminations, microbial toxins or oncogenic sequences, relatively high protein
yield and scalability. In 1990‟s, the widely acknowledge advantages of plant based
molecular farming were not sufficient to displace the conventional fermentor based
plantforms. The early technical limitations in terms of low yield, low recovery during
processing and lack of regulatory framework for plant based pharmaceuticals (Ma et al.,
2003) were the major hurdles. The recent development by several research groups and
industry-led consortia has eliminated these obstacles by different approaches for higher
accumulation and efficient downstream processes. Good manufacturing practices (GMP)
are now available for plant based system and the first products are entering clinical trials
(Fischer et al., 2012).
Different plant species and tissues have now been used as production system for a variety
of recombinant proteins including cereals, legumes, leafy crops, oilseeds, higher plant
tissue and cell culture, fruits, vegetables, algae, moss and higher aquatic plants (Stoger et
al., 2005). All of these production hosts have their own merits and demerits for protein
production in terms of time required, cost of end product, safety, scalability, ease of
transformation, downstream processing, protein folding and transportation.
2
I.2. The seed
Seeds are the dispersal and propagation units that allow plants to spread out and grow in
new territories, and help plants to survive under unfavourable conditions in a dormant
stage. A seed is a small embryogenic plant emerged from an ovule after fertilization and it
is composed of three main parts: a seed coat, an endosperm and an embryo with one or
two cotyledons (Fig. I.1D). Structural division of plant seed include: monocotyledonous
seeds (cereals), endospermic dicotyledonous seeds (tobacco) and non-endospermic
dicotyledonous seeds (beans). In seed, the embryo is surrounded by endosperm cells
which supply nutrient to embryonic tissues. The entire seed tissues are provided by seed
coat that acts as a mechanical barrier to protect the seed and to remain dormant for a
longer time (Khan et al., 2012).
The endosperm is well recognized for its importance as food and feed. Cereal seed
endosperm contains 70% starch in terms of dry weight. Starch is made of glucan
polymers, amylose and amylopectin, that are packed into semi-crystalline granules in
amyloplasts (James et al., 2003). Cereals are the main protein provider in certain regions
where rice or maize is the main staple food (Shewry, 2002). In cereals, endosperm
provides major space for for storage proteins, which are responsible for the visco-elastic
properties of the dough. Hence in the case of wheat the seed protein content is a major
quality determinant for bread, pasta and other backed goods.
Figure I.1 Illustration of the seed anatomy of three plant models. A. Cross section of Nicotiana
tabacum.sc. seed coat, cot. cotyledon, en. endosperm (Tomlinson et al. 2004). B. Light microscopy with
toluidine blue of Medicago truncatula mature seed (non-endospermic dicotyledonous) rad. Radical, mes.
Mesophyll, col. cotyledon, en. endosperm (Figure from Prof. Eva Stoger Lab at Vienna, Austria) C. Cross
section of wheat seed (monocotyledonous) sc. Seed coat, en. endosperm, cot. cotyledon (Figure from Prof.
Eva Stoger Lab at Vienna, Austria) D. Longitudinal section of wheat grain (Figure adapted from Wikipedia;
Wugo).
3
I.2.1. Seed storage proteins
Seeds are rich reservoirs of storage proteins (seed storage proteins, SSPs), which
constitute up to 90% of the total protein fraction in developing seeds and are crucial for
germinating seedlings providing them with carbon, nitrogen and sulphur (Kumamaru et
al., 2007). The total mass of seed proteins varies from 10-15% of grain dry weight in
cereals to up to 45% (dry weight) in legumes.
Seed proteins are classified into three types i.e. storage protiens, structural and metabolic
proteins and protective proteins. SSPs occur in three different tissues of the grain (embryo,
aleurone layer and endosperm) and can be divided into different classes (Galili 2004). A
range of criteria have been used to define and classify seed proteins. T. B. Osborne
(Shewry 2002) developed a classification based on solubility of plant proteins known as
“Osborne fractionation” which is still used today, especially by cereal scientists. Thus,
four groups are defined: water soluble albumins (found mainly in dicots), salt soluble
globulins (found both in dicots and monocots), alcohol soluble prolamins (found
exclusively in cereals) and glutelins (soluble in dilute acids, found also in cereals). Cereal
seed proteins can also be classified on the basis of their metabolic properties, deposition
sites and allergenic properties, but the latter is not so often in use (Breiteneder et al. 2005).
I.2.1.1. Prolamins
Prolamins are the major endosperm storage proteins in cereals with the exception of oats
and rice. They were originally classified as soluble in alcohol/water mixtures but some
occur in alcohol-insoluble polymers. All individual prolamins are soluble in alcohol in the
reduced state. However, variations in their structure are the clue of distinct evolutionary
origins of the Triticeae (wheat, barley and rye) and the Panicoideae (maize, sorghum,
millets). The prolamin superfamily has been reorganised in subgroups according to
structural and evolutionary relationships. On the broad spectrum, sequence similarities can
be found among different cereal prolamins. Based on sequence repetition, the S-rich and
S-poor groups are relatively similar as they both contain proline and glutamine rich
sequences. However, similar motifs can also be found in non-repetitive domains. These
similarities suggest the common evolutionary origin of S-rich and HMW prolamins
(Shewry et al., 1995). The cereal prolamin super-family has been defined on the bases of
4
wider comparison of evolutionary and structural similarities of several groups of maize,
oats and rice prolamins, 2S albumin storage proteins and other seed proteins (Kreis et al.,
1985).
The prolamins of maize (called zeins) constitute more than half of total seed proteins
synthesized within endosperm tissue. Alpha-zeins have a relative molecular mass of 19
and 22 kDa and constitute the most important class of zeins, comprising 70% of the total
prolamins. Alpha-zeins do not appear to be related to any other prolamins except the
alpha-type of the other panicoid cereals. The remaining three classes (beta, gamma and
delta) are sulphur rich and contrary to alpha-zeins they have higher cysteine content and
form polymers (Kumamaru et al 2007; Shewry and Halford 2002; Woo et al. 2001,).
I.3. Seed as bioreactor
Plant seed is an attractive host for molecular farming because recombinant protein can be
targeted to naturally available storage organelles allowing the stable accumulation of
recombinant proteins in endosperm and embryo tissues. Seed tissues are gifted with
molecular chaperones and disulfide isomerases to facilitate folding and assembly of
proteins. As well, the endosperm and embryo tissues are desiccated to prevent proteolytic
degradation and can therefore remain stable for years (Ramessar et al., 2008). Seed from
various plants has been considered as production host for large number of heterologous
proteins. Seed crops such as: wheat (Arcalis et al., 2004; Stoger et al., 2000), rice (He et
al., 2011), barley (Schunman et al., 2002), arabidopsis (Downing et al., 2006), maize
(Ramessar et al., 2008; Rademacher et al., 2008) brassica (Parmenter et al., 1995), pea
(Perrin et al., 2000), safflower (Nykiforuk et al., 2010), tobacco (Floss et al., 2008) and
soybean (Cunha et al., 2010) have been used as bioreactor for different recombinant
proteins. Cereal crops such as maize, rice, barley and wheat have been used as hosts for
recombinant proteins (Table 2) and the former three of these are being developed
commercially (Stoger et al., 2002a). The structure of seeds provides advantages in terms of
post-harvest processing because the small and homogenous size of seeds is beneficial for
the concentration and downstream processing of recombinant proteins. Protein targeting is
one of the crucial factors influencing protein yield and stability in seeds which also
determine posttranslational modification and accumulation levels (Ramessar et al., 2008).
5
I.3.1. Wheat
Wheat is the major crop grown all over the world. It is the most widely consumed food
crop because of its adaptability and high yield in a wide range of environments. Wheat was
the first Triticeae species to be stably transformed (Vasil et al. 1992). Among cereals,
wheat has been used rarely as a production host for recombinant proteins because the gene
transfer in wheat is less efficient and requires more specialized skills for stable
transformation (Stoger et al. 2000). Wheat is an attractive candidate for molecular farming
because of its higher protein content (>12%) and lower production cost. Different
ecombinant proteins have been expressed in wheat plant (Arcalis et al, 2004, Stoger et al.,
2000). This is important to mention that the antibodies expressed in wheat seed have
remain stable for several years at room temperature without any detectable loss of activity
(Stoger et al., 2000). Wheat is a self-pollinating crop, which implies that the chances of
out-crossing to non-transgenic crops and wild relatives are minimized. To make wheat
more feasible for molecular farming, more efficient gene transfer and regeneration
technology is needed. This may be accomplished through the development of routine
methods for high-frequency stable transformation i.e by Agrobacterium-mediated gene
transfer. Attempts to express different proteins in wheat seeds have so far produced lower
yields of recombinant proteins compared to other cereal species.
I.4. Subcellular protein targeting
The stability and accumulation of the recombinant proteins can be achieved by targeting to
appropriate subcellular compartment (Streatfield et al., 2007). The targeting organelle is
not the same for all the recombinant proteins because of their diverse structures and
properties. Literature provides evidences in terms of appropriate targeting approaches for
particular types of proteins, e.g. a range of recombinant antibodies expressed in plants.
Proper folding, assembly, the formation of disulfide bonds and glycosylation influence the
stability and functionality of antibodies, therefore targeting of such polypeptides to the
secretory pathway is recommended. Secretion of proteins to the apoplast is achieved by
the addition of N-terminal signal peptide to each antibody chain. An additional C-terminal
KDEL or HDEL tag can also reclaim secreted proteins to the endoplasmic reticulum
(Conrad et al., 1998).
6
Plant seed storage tissues contain more complex endomembrane system than non-seed
cells where more complex protein can travel through storage proteins as their final
destination. The plant endomembrane system involves a series of functionally-specialized
membrane-bound compartments and organelles including an abundant ER reminiscent of
mammalian secretory cells, ER-derived protein bodies, endosomes, the Golgi apparatus
and different types of vacuoles. Storage proteins in cereal endosperm accumulate in
different compartments, and their abundance and distribution varies according to the
species (Arcalis et al., 2004). Proteins with N-terminal signal peptides are translocated
into the ER and are normally secreted to the apoplast if there are no further targeting
signals, or retrieved to the ER if a C-terminal H/KDEL tetrapeptide is present. In addition
to ER lumen, seed tissues evolve storage organelles such as ER derived protein bodies
(PB), protein storage vacuoles (PSVs), starch granules and oil bodies. In different studies,
these naturally available organelles have been utilized as final destination for recombinant
proteins to achieve higher accumulation, stability, and facilitate recovery (Khan et al.,
2012). Targeting recombinant proteins to protein bodies provide additional benefits such
as increasing the efficacy of oral vaccine delivery (Takagi et al., 2010).
I.4.1. Endoplasmic reticulum
The endoplasmic reticulum (ER) is considered as the “gateway to the secretory pathway”
(Ibl and Stoger 2011). The endoplasmic reticulum (ER) is a part of the plant
endomembrane system with highly conserved functions in lower and higher plants. The
endoplasmic reticulum is crucial for secretory proteins as they are synthesized on the
rough ER and are subjected to the machinery for their processing including glycosylation,
disulfide bond formation, proper folding and oligomerization (Hadlington and Denecke
2000). For accumulation in the ER and in ER-derived storage organelles, the constructs
are designed to contain a targeting sequence for ER retention such as the C-terminal
tetrapeptide H/KDEL, which prevents protein secretion (Takaiwa et al., 2007).
Recombinant proteins have shown higher accumulation and stability when targeted to ER
in various plant species. Interestingly, heterologous proteins which contain a KDEL signal
and accumulate in the ER of tobacco leaves, tend to secreted or accumulated in protein
storage vacuoles in seeds (Petruccelli et al., 2006). In tobacco leaves, the single-chain
7
variable fragment (scFv)-KDEL antibody is expressed with accumulation levels of 1.65%
TSP (Fischer et al., 1999). The fusion of hydrophobins HFBI sequence from Trichoderma
reesei with Green fluorescent (GFP-HFBI) has successfully induced the formation of
protein bodies with higher concentration to 51% (TSP) of fusion proteins in tobacco leaves
(Fig. I.2; Joensuu et al., 2010). In another study, the expression and proper accumulation of
two protein components of KDEL-tagged spider dragline silk in tobacco leaves has also
been described (Menassa et al., 2004). In seeds, an antibody scFv-Fc construct fused to
KDEL was produced up to 12% TSP in Arabidopsis where the heterologous protein was
detected in the periplasmic space but absent from the ER. In this case the presence of
heterologous protein apparently disturbed the ER retention and leading partial secretion of
the recombinant protein and endogenous storage proteins in seeds (Van Droogenbroeck et
al., 2007). Similarly, an unexpected deposition of KDEL-tagged human serum albumin
protein was observed in wheat endosperm, where the recombinant protein was deposited
together with prolamin aggregates within the storage vacuole (Arcalis et al., 2004). This
mistargeting of KDEL-tagged proteins may be a result of the unique storage properties of
seed tissues. Strong over-expression of GFP without fusion partner in transient N.
benthamiana leaves induced the formation of necrotic lesions, In contrast, when GFP is
expressed as a fusion with HFBI, an increase in the expression up to 2 fold was observed
and infiltrated leaves remained healthy up to 10 dpi (Joensuu et al., 2010).
Figure I.2 ER-targeted Green fluorescent protein (GFP). Confocal image of the ER targeted recombinant
protein (GFP) expression shows the ER network indicated by the flourecent protein (Joenssu et al., 2010).
8
I.4.2. Protein bodies and protein storage vacuoles
Cereal endosperm includes two types of protein storage organelles: protein bodies, which
are derived from the ER, and protein storage vacuoles (PSVs), which are formed de novo
(Jiang et al., 2001). In cereals, all storage proteins are synthesized on the surface of the
ER. After synthesis, they are translocated from the cytoplasmic side of the rER to the ER
lumen. The newly formed polypeptides are then assembled into protein bodies accretions.
Protein bodies either remain in ER or they may deposit through endomembrane system to
protein storage vacuoles (PSVs; Vitale and Denecke, 1999). The protein bodies in cereals
are surrounded by a membrane of ER origin. In cereals, seed storage proteins are
transported to PSVs in three different pathways. These include the transport through
Golgi-derived dense vesicles, precursor-accumulating (PAC) vesicles and via an
autophagy-like process (Tosi et al., 2009).
As shown in wheat and barley, protein bodies containing prolamins are deposited into
central large PSV by an autophagy-like process (Levanony et al., 1992; Herman et al,
1999). In other cereals, such as maize and rice, the protein bodies are synthesized within
ER lumen and they remain in these organelles. Rice storage proteins are deposited into
two different populations of protein bodies, the regular shape and ER origin protein bodies
(PB-I) and irregular vacuolar PB-II. In cereals, the origin and exact mechanism of protein
bodies synthesis is still unclear. The synthesis and trafficking of storage proteins in
different cereals has been discussed further in section I.5.
In molecular farming, both these compartments i.e protein storage vacuoles (Frigerio et al.
2008; Vitale and Hinz 2005) and protein bodies (Coleman et al. 1996; Conley et al., 2009)
have been used to accumulate variety of recombinant proteins (Takaiwa et al., 2007). The
targeting of recombinant proteins to ER derived protein bodies and PSVs have resulted in
stable and higher accumulation (Arcalis et al., 2004; Galili 2004; Herman and Larkins
1999; Levanony et al., 1992).
I.4.3. Oil bodies
Lipid particles are found in seeds such as sunflower, safflower, rapeseed and mustard. Oil
seeds save lipids in subcellular particles as food reserves in form of triacylglycerol (TAG)
9
used to supply energy for germination and post-germinative growth. The TAGs are present
in small subcellular spherical oil bodies, being approximately 0.5-1 µm in diameter.
Recombinant proteins targeted to oil bodies are relatively stable, do not aggregate or
coalesce and are surrounded by a protected layer of unique proteins called oleosins (Huang
1996). The recognition signals are provided by oleosins for lipase binding during oil
mobilization in seedlings (Huang, 1996; Murphy, 1993).
Oil bodies are used as storage organelles in plant biotechnology because of their presence
in various plant tissues. Oilseeds in particular are exploited for protein expression. Oil
body targeting has its own advantages in terms of purification of fused proteins due to a
high ratio of triacylglycerides. Using the oleosin-fusion system, the pharmaceutical protein
hirudin has been successfully expressed in Brassica species i.e. Brassica napus (Boothe et
al., 1997) and Brassica carinata (Chaudhary et al., 1998). Recently, recombinant protein
Apoliprotein Al Milano was expressed as a fusion protein in transgenic safflower seed
with high levels of expression corresponding to 7g of ApoAlmilano per kilogram of seed
in the transgenic line selected for commercialization (Nykiforuk et al., 2011). One of the
molecular farming product is recombinant human insulin expressed in safflower seeds is in
phase III clinical trials and closest to the market (Fischer et al., 2011).
I.4.4. Starch binding domains
Starch is the primary energy storage polysaccharide in all plants, a main source of calories
in the human diet (James et al., 2003) and widely used for food and industrial purposes.
Starch is composed of α-1,4-linked glucose residues organised into the essentially linear
amylose and the branched amylopectin containing α-1,6-linkages. Starches are deposited
as semi-crystalline granules in chloroplasts of leaves (transitory starch) and in amyloplasts
of storage organs. Starch consists usually of 20-30% amylase and 70-80% amylopectin (Ji
et al., 2003). Starch granule size varies from less than 1 μm to over 100 μm with spherical
or elongated shapes (Smith 2001). Cereals such as wheat and barley contain a mixture of
small and large granules known as A and B granules (Fig. I.3), which differ in their
morphology and chemical composition (Ao and Jane 2007 and Geera et al., 2006). In
wheat, A-granules are between 10 and 38 μm in size and disc- or lenticular-shaped, while
B-granules are smaller than 10 μm and possess a spherical or polygonal morphology
(Wilson et al., 2006).
10
Starch-binding domains (SBD) derived from bacterial enzymes that catabolise starches
have the capability to bind two helices of starch and thus potentially useful for targeting
recombinant proteins to starches. SBDs independently retain their function even if fused to
another protein and can therefore be used to target recombinant proteins to existing starch
granules (Janecek 1999).
Figure I.3 Wheat A-type and B-type granule structure. (Buléon et al., 1998)
I.5. Seed storage protein trafficking
In the seed, storage proteins move along specific routes within the endomembrane system.
Seed cells produce vast amounts of SSPs with different subcellular destinations, including
complex protein storage vacuoles and protein bodies derived from the ER (Ibl and Stoger,
2011). Storage proteins, such as albumin and globulins, start the journey from ER lumen
and are then transported to PSVs, passing through the Golgi apparatus via dense vesicles
(Hohl et al., 1996). In contrast to this pathway, 2S albumins and 11S globulins in pumpkin
seed accumulate in PSVs, bypassing the Golgi apparatus via precursor-accumulating
(PAC) vesicles (Hara-Nishirama et al., 1998).
Storage protein trafficking has been extensively studied because of the nutritional value of
such proteins. In cereals, globulins follow the same routes as described above: they
accumulate in the cell‟s PSVs passing through the Golgi apparatus. The deposition of
prolamins in cereal endosperm is specific to the species. In cereals such as rice, maize and
sorghum, prolamins aggregate into dense protein bodies within the rough-ER lumen (Fig.
1.4) and remain attached to this organelle (Muench et al., 2000). The protein bodies are
encapsulated by a membrane protecting them from proteases and desiccation during seed
development, until they are used up as an energy source for germinating seedlings (Muntz
1998). Thus, protein bodies are particularly favoured for deposition and storage of large
11
amounts of recombinant proteins which are poorly secreted or toxic to the host (Torrent et
al., 2009). Prolamins are exclusively found in cereal endosperm. In wheat and oat, the
prolamins accumulate to form aggregates and bud off the ER as in other cereals. But later
they are incorporated into PSVs by an autophagy-like process bypassing the conserved
mechanism of the Golgi mediated targeting (Arcalis et al., 2004; Galili et al., 1993;
Levanony et al., 1992). The role of the Golgi apparatus has been controversial but
generally, the Golgi independent pathway is more widely accepted, although a Golgi-
dependent pathway has also been proven for small amounts of prolamins (Miflin et al.,
1981).
Wheat endosperm contains two types of PBs: the low density type protein bodies (light
PBs) where gliadins are present, and the high density type of protein bodies (dense PB),
where high molecular weight glutenins are located. Both the high molecular weight
glutenins and gliadins are transported and deposited to the PSV by two separate processes,
the Golgi-dependent and the Golgi-independent pathway (Kumamaru 2007). Wheat
protein bodies do not remain as separate cytosolic structures but are sequestered into
provacuoles (Rubin et al., 1992), predominantly in the cells of the subaleurone layer (Fig.
I.5A). Surprisingly, cereal prolamins do not contain any known ER or vacuolar targeting
signals. Wheat protein bodies are sequestered within a large, central vacuole. In wheat,
gliadine and glutenins are the major classes of prolamins in the endosperm, but also
contains an 11S globulin homolg and triticin as minor storage proteins, which account for
5% of total seed proteins (Singh et al., 1991). Gliadins accumulate in protein bodies as
monomers, while glutenins assemble via non-covalent interactions and intermolecular
disulfide bonds. Unlike prolamins, gliadins contain N-terminal signal sequences that
determine their passage into lumen of the ER. However, it is not known why some gluten
proteins pass and others bypass the Golgi apparatus (Tosi et al., 2009).
Zein constitutes more than half of the total seed proteins in maize endosperm (Fig I.3B).
Different classes of zeins can be found in endosperm 10-40 days after pollination (DAP)
and constitute approximately 50% of the total proteins in mature seed (Lending and
Larkins 1989). Maize γ-zein is able to induce protein body formation in dicot tissues
regardless of the presence of other zein subunits (Coleman et al., 1996). -Zein is
composed of four domains: eight repetitions of the PPPVHL motif, a hydrophobic cystein
12
rich C-terminal sequence, Pro-X enclosing proline residues and a 19 amino acid signal
peptide (Prat et al., 1985). In maize endosperm tissues, zeins located in 2 µm spherical
protein bodies within the ER lumen where γ-zeins are located at the periphery of the dense
bodies. N-terminal proline rich sequences contribute to protein body formation and its
retention within the ER lumen. Three types of zein (β, γ, δ) have been successfully
produced in leaves of transgenic arabidopsis, where they are deposited to ER derived
protein bodies (Bagga et al., 1995, 1997).
Alpha zeins are composed of two major subclasses (19 and 22 kDa zeins), and constitute
the most important class of zeins, accounting for 70% of the total prolamins. The
remaining minor group of three zein classes include delta, beta and gamma (δ, β and γ)
having molecular masses of 10, 15 and 27 kDa respectively, when separated on SDS-
PAGE. -zeins are rich in cystein while both δ and β are rich in methionine residues. Beta
zeins share some features with γ-zein, such as both being sulphur rich, carrying conserved
N and C terminal peptides and requiring a reducing agent for their solubility in alcohol.
Rice endosperm accumulates three types of storage proteins, the glutelins, prolamins and
α-globulins (Muench et al., 1999), where globulins (known as glutelins) and prolamins are
in majorityand make up 60-80% and 20-30% of total seed proteins, respectively
(Kawakatsu et al., 2010). These storage proteins are transported to different sites of the
endomembrane system (Krishnan et al., 1986, Tanaka et al., 1980). In rice seeds, more
than 80% of the total seed proteins are deposited as storage proteins in protein bodies as
protein storage organelles. There are two distinct protein bodies found in the rice
endosperm known as PB-I, which originates from the ER and contains prolamins, and
vacuolar PB-II, accumulating globulins and glutelins (Fig. I.6; Krishnan et al., 1986;
Yamagata and Tanaka 1986). In rice, the chaperone BiP is closely associated with
prolamins and is located at the periphery of rice prolamin bodies, which aids the deposition
of prolamins on the surface of protein bodies (Li et al., 1993; Muench et al., 1997). BiP is
known to be present in cereal PBs, where it is considered to play a role in storage, protein
folding and assembly (Li et al., 1993). Okita and co-workers could show that prolamin
mRNA plays a crucial role in the deposition of prolamins within the rice endosperm.
Evidence has been found that rice prolamins and globulins/glutelins are translated on two
separate sub-domains of ER. Prolamin mRNAs are targeted to ER membrane surrounding
13
the protein bodies by a mRNA signal recognition process (Hamada et al., 2003a; Hamada
2003b), whereas mRNA localization on the cisternal ER membrane results in
globulin/glutelin transport to the PSV (Li et al., 1993a). The specific mRNA trafficking to
protein bodies appears to involve the cytoskeleton (Hamada et al., 2003b).
Figure I.4 Conceptual diagram of the ontogeny of PBs and PSVs. In cereals, protein bodies are form
within the rER. After synthesis, they can either bud off the ER, remain in the cytoplasm or be sequestered
into protein storage vacuoles. PSVs are formed as the result of ER-synthesized storage proteins progressing
through the endomembrane secretory system to the vacuole for accumulation (Hermans and Larkins 1999).
Figure I.5 Deposition of wheat protein bodies. Light microscopy. Cross section of a wild type wheat seed
stained with methylene blue. The protein bodies are deposited within the large central vacuole (arrow) in the
wheat endosperm cells. Figure adapted from Prof. Stoger‟s Lab at Vienna, Austria.
14
Figure I.6 Protein bodies deposition in maize and rice. Light microscopy. Semi-thin cross section of wild
type maize seed treated with methylene blue. Protein bodies (arrow) are distributed in first few cell layers of
maize endosperm tissues. Rice. Light microscopy. Methylene blue stained semi-thin section. Protein bodies
Type I and II (arrow) are visible within rice endosperm. Figure adapted from Prof. Stoger‟s Lab at Vienna,
Austria.
I.6. Fluorescent protein markers
Transgenic plants are generally developed through co-insertion of selectable marker genes
together with the transgenes. Selection with antibiotics or herbicides is then used to enrich
for plants with successful integration of the transgenes in the host cells. In contrast to
selectable marker genes, fluorescent reporter genes (GUS; GFP, dsRed) can be easily
detected by phenotypic analysis (Miki and McHugh 2004). For the identification of
transformed cells, which are very few within a large background of non-transformed cells,
reporter genes (cat, uidA, luc, gfp, dsRed) are favoured for plant transformation constructs.
To monitor the gene expression, protein localization and intracellular protein trafficking
may be visualized with protein markers in situ, without harming the plants (Ziemienowicz
2001). GFP and DsRED are favoured because of their high quantum yields and stability in
living cells.
Fluorescence is a phenomenon in which a material absorbs light of one colour
(wavelength) and emits light of a different colour (wavelength). Fluorescent reporter
proteins, such as green fluorescent protein (GFP) and red fluorescent protein (DsRED), in
plant cell biology are essential to track, visualise and quantify gene expression in the host
cells. Monitoring of CAT, GUS and LUC activity requires the sample preparation (Jach et
al., 2001). Due to intrinsic fluorescence detection, the fluorescent protein offers an
advantage over GUS and luciferase marker proteins with respect to being used without any
15
additional specific substrates to monitor gene expression and analysis of transformants in
the first generation.
I.6.1. Discosoma red fluorescent protein
Red fluorescent protein (DsRed) has been isolated from coral of the genus Discosoma
(Matz et al., 1999) and is used as natural red chromophore. DsRED has the maximum
excitation and emission shifted to the red when compared to GFP (excitation/emission
maximum of GFPs 488–495 nm/507–510 nm versus 558 nm/583 nm for DsRED).
Discrimination of DsRED and GFP fluorescence can be easily done by using appropriate
filter settings; allowing simultaneous multicolour imaging of different genes (Jach et al.,
2001). DsRed is commonly utilized to examine developmental and spatial gene expression
in transgenic plants. Like GFP, it does not require the preparation of protein extracts,
additional substrates or enzymes. DsRed allows non-invasive, non-destructive detection at
very early stages of plant transformation. DsRed was first expressed in tobacco BY2
protoplast cells by Mas et al. (2000). The DsRed is visualized by the red phenotype of
seeds, seedlings and plant lines, which express the protein in the cytosol, ER or
chloroplast. The expression of red fluorescent proteins does not affect the fertility and
germination of seeds (Jach et al., 2001). Red fluorescent marker proteins are traceable
markers and can be easily detected by simple microscopic analysis. The fused protein can
be easily detected at very early stages of its development using low tech equipment.
Figure I.7 Red Fluorescent protein as selection marker. Detection of transgenic seeds on a segregating
ear of maize using red fluorescent protein (DsRed) as a visible marker (Rademacher et al., 2009)
16
I.7. Encapsulation of recombinant proteins
Several strategies have been exploited in an attempt to maximize heterologous protein
accumulation in plant cells. Despite successful alternative expression systems, foreign
protein expression levels are relatively lower in the plant expression system due to major
challenges, which include insufficient accumulation levels and lack of efficient purification
methods for recovery (Joennsu et al., 2010). Expressing recombinant proteins as fusions to
protein - stabilizing partners has a positive impact on the protein accumulation and
purification, and efficient and cost-effective purification of recombinant proteins from
plants (Witte et al., 2004). To enhance recombinant protein accumulation, a variety of
fusion proteins has been used in plants such as cholera toxin B subunit (Molina et al.,
2004), viral coat proteins (Canizares et al., 2005), ubiquitin (Mishra et al., 2006), β-
glucuronidase (Dus Santos et al., 2002), human immunoglobulin (IgG) α-chains (Obregon
et al., 2006). To enhance the purification process, recombinant proteins are fused affinity
tags such as His-tag, glutathione S-transferase-tag, FLAG-tag, c-myc-tag, Arg-tag,
calmodulin-binding peptide, maltose-binding protein, the cellulose-binding domain (Lichty
et al., 2005) and eight-amino acid StrepII epitope tag (Skerra and Schmidt 2000).
However, these tags include costly chromatography techniques for protein purification that
is difficult to scale up for industrial use (Menkhaus et al., 2004; Waugh 2005).
Large-scale partitioning of different biological components, including recombinant
proteins, is often a bottleneck in modern biotechnology. To increase the accumulation of
valuable recombinant proteins in seeds, and to facilitate protein purification, the properties
of seed storage proteins and/or the trafficking pathways that lead to storage protein
deposition in seeds have been exploited (Table I.1). Therefore, different fusion based
strategies have been developed to deal with these issues (Terpe 2003) including, zeins,
ELPs and Hydrophobins.
17
Figure I.8 Using storage organelles for the accumulation and encapsulation of recombinant proteins
(Khan et al., 2012).
Table I.1 Fusion partners for creation of artificial storage organelles.
I.7.1. Zein
In the cereal endosperm, zeins are accumulating in the endoplasmic reticulum-derived
protein bodies and can accumulate up to 15% of total endospermic proteins (Marzbal
1998). Zeins are among well studied seed storage proteins in maize. Endospermic cells of
maize, together with other zein classes (α, β, γ, δ) form large complexes known as protein
bodies in endoplasmic reticulum lumen (Fig. I.9). No specific canonical HDEL/KDEL
retention/retrieval signals responsible for retaining proteins in the ER have been found in
Origin Purification Method Increased yield PB-Formation
Zein Plant Isopycnic sucrose density
centrifugation
15-100 fold Protein repeat domain,
Pro-X domain,
Cystein domain
Elastin-like
polypeptides
Animal
Inverse transition cycling
(ITC)
2-100 fold
VPGXG
Hydrophobin Fungus Surfactant-based aqueous
two phase system (ATPS)
Unknown Hydrophobic
interation.
8 cysteines
18
γ-zeins. This phenomenon of protein retention and localization within the ER of
endosperm of maize is still to be discovered.
Protein fused to N-terminal proline–rich domain of γ-zein, known as Zera has capability to
stably express recombinant proteins in plants and have induced an increase in overall
yields (Mainieri et al., 2004; Torrent et al., 2009;). Zera fusions with other protein are able
to self-assemble and accumulate the recombinant proteins inside protein bodies. The exact
synthesis of protein bodies is still undetermined, but it has been proven in earlier studies
that proline rich domains made of hexapeptide PPPVHL are responsible for retention of
proteins in the ER. There is evidence that β-zein sharing conserved N- and C-terminals
with γ-zeins, which are then capable of inducing protein body formations (Colemann and
Larkins 1999). However, expression of only α-zeins fails to induce protein body structures
when expressed into different hosts.
Figure I.9 Localization of storage proteins in maize endosperm. Fluorescence microscopy. Two classes
of Zein (α, γ) expressed in maize leaves. Figure adapted from Prof. Stoger‟s Lab at Vienna, Austria.
I.7.2. Elastin-like polypeptide
Elastin like polypeptides (ELP) are synthetic biopolymers made of repeats of amino acid
`Val-Pro-Gly-Xaa-Gly` sequences, where residue Xaa is any amino acid except proline
(Urry, 1988). Elastin-like polypeptides (ELP) are found in all mammalian elastin proteins
and can be purified with temperature based non chromatographic methods. Elastin-like
polypeptide (ELP) tags upon expression with particular proteins induce protein body
formation of the same size and morphology to natural protein bodies found in maize
endosperm. This has been demonstrated by ELP-GFP fusion in tobacco leave which
19
positively affected the stability and resulted in higher accumulation of recombinant
proteins (Fig. I.10; Conley et al., 2009).
ELP fused protein can be purified with inverse transition cycling (ITC) which is a fast,
simple, easily scalable and cheap non-chromatographic method for protein purification.
This method is used for purification of antibodies, cytokins and spider milk proteins from
transgenic plants. When heated above transition temperature, ELP are converted to
hydrophobic aggregates and form β-spiral structures. This thermal response property has
also been transferred when ELPs are fused with recombinant proteins, enabling the
purification through ITC (Conley et al., 2009). This heat sensitivity is directly connected to
the size of the proteins in plants, such as that the transition temperature could be 30-39 C
for proteins ranging from 30-65 kD in mass (Stibora et al., 2003). However, the heat
sensitivity is inversely proportional to the size in E.coli (Meyer and Chilkoti 1999). ELP
fusion has found 40 times more abundant (compared to control) when expressed in tobacco
seeds (Scheller et al., 2006).
The proteins, once fused with the ELP tags, exhibit an increase in accumulation in plants,
but this procedure still needs to be optimized in order to maximize the accumulation levels
of these fused proteins in plants. ELP fusion proteins have significantly accelerated protein
accumulation and purification in plants, but the impact of ELP fusion proteins on overall
factors defining protein quality still needs to be determined.
Figure I.10 Fluorescence microscopic image of tobacco leaf. In the presence of an ELP fusion tag, the
ER-targeted GFP was detected in brightly fluorescing spherical-shaped particles distributed throughout the
cells of the leaf (Colney et al., 2009)
20
I.7.3. Hydrophobins
Hydrophobin fusions are originally developed for the purpose of purifying proteins from
fungal cultures supernatant (Conley et al., 2011). It has already been demonstrated that
hydrophobin fusion can induce higher accumulation of target proteins in plants and fungi
(Linder et al., 2004). Endoplasmic reticulum-targeted hydrophobins induce the formation
of novel protein bodies (Table.I.1; PBs; Conley et al., 2009b; Torrent et al., 2009).
Purification of macromolecules is the key factor in alteration of the fusion protein partners,
through surfactant-based aqueous two-phase system (ATPS; Linder et al., 2004). ATPS is
a simple, rapid, and inexpensive procedure that can result fast separations (Persson et al.,
1999). At industrial points of view, hydrophobins are attractive because they require a one-
step purification procedure and can be easily scaled up for industrially valuable proteins
(Linder et al., 2004; Selber et al., 2004).
Figure I.11 ER-targeted GFP Hydrophobin fusion (GFP-HFBI) promotes the formation of PBs in
Nicotiana benthamiana. Leaf epidermal cell accumulation GFP-HFBI fusion protein in protein bodies
(Joensuu et al., 2010).
21
Table I.2 Examples of recombinant proteins produced in cereal seeds.
TSP: Total Seed Protein; RSP: Rice Seed Powder
Crop Recombinant protein Plant Tissue/ Subcellular
Localization
Expression
Level/Yield
Promoter Reference
Maize
Barley
HIV 2G12
HIV 2G12SEKDEL
Avidin
Escherichia coli heat
labile enterotoxin (LT-B)
HIV Diagnostic Reagent
(HIVDR)
Endosperm/embryo
ER-derived Protein Bodies
Endoplasmic Reticulum (ER)
Starch granules
Endoplasmic reticulum
38-75 µg per gram
30μg/g dry weight
5.7% TSP
1.3g/kg
150 µg of reagent g-1
Endosperm-specific rice glutelin-1
glutelin-1 (gt-1) promoter
Maize ubiquitin promoter
27-kDa γ-zein promoter
B×17 promoter
Ramessar et al., 2008
Rademacher et al., 2008
Kusnadi et al., 1998
Chikwamba et al., 2003
Schünmann et al., 2002
Rice
Wheat
Human serum albumin
(HSA)
7Crp peptide
Human interleukin
Cholera toxin B subunit
Human Serum Albumin
Single-chain Fv antibody
(ScFvT84.66)
Endosperm
ER-derived Protein Bodies
Endoplasmic reticulum
Protein bodies (PB) I & II
ER-derived Protein Bodies
Endosperm
2.75g/kg of Rice
60 µg/ grain
4.5mg/100g RSP
30 µg/g CBT per Seed
0.5% TSP
30μg/g
Endosperm-specific promoter,
Gt13a promoter,
Glutelin GluB-1
Glutelin GluB-1
Ubiquitin-1 promoter
Maize ubiquitin promoter
He et al., 2011
Takaiwa et al., 2009
Fujiwara et al., 2010
Nochi et al., 2007
Arcalis et al., 2004
Stoger et al., 2000
22
Objectives of the study
In the context of molecular farming, plant seeds are ideal vehicles for the production of
recombinant proteins because they contain specialized compartments for the stable
accumulation of storage proteins. Recombinant proteins and endogenous storage proteins
in dry seeds each benefit from the same stable environment, allowing long-term storage
and batch processing. Seeds contain several unique types of storage organelles to which
recombinant proteins can be directed and in which they accumulate. These include protein
bodies derived from the endoplasmic reticulum (ER) and protein storage vacuoles. The
maize seed storage protein γ-zein is an endogenous prolamin that can trigger the formation
of protein bodies both in the seed and non-seed tissues of dicotyledonous plant species. In
this project, the repetitive (PPPVHL)8 and PX domains from γ-zein protein were
expressed as a fusion with the red fluorescent marker protein DsRed to allow the behavior
of the fusion protein to be characterized visually in different wheat tissues.
The main objective of this thesis was to study the expression of recombinant wheat plant
and to study trafficking and deposition of the model fusion protein in different wheat
tissues, such as the endosperm, embryo and leaves, to investigate its ability to form protein
bodies for the storage of recombinant fusion proteins in diverse environments. Our
hypothesis was that in wheat cells the model γ-zein-DsRed fusion protein would form a
unique and separate population of protein bodies budding from the ER into the cytoplasm,
but because endogenous prolamins would be present in the endosperm it would not be
possible to predict the behavior of the model protein in this setting. We therefore
investigated the precise localization of the fusion protein to find out whether it would form
a separate population of ER-derived protein bodies or mix with endogenous prolamins in
endogenous glutelin bodies. Autophagy-like processes are well-documented in wheat
endosperm cells, so we also set out determine whether the model protein was ultimately
incorporated in the protein storage vacuole like wheat glutenins.
One of the bottlenecks in molecular farming is the low yields of recombinant proteins in
transgenic plants, often reflecting a combination of low-level accumulation and inefficient
extraction and downstream processing. We used the model fusion protein γ-zein-DsRed to
investigate strategies for the improvement of recombinant protein accumulation in wheat,
23
based on preventing exposure to proteolytic enzymes in the cytosol. This thesis
contributes to the body of knowledge on recombinant protein expression in plants by
studying the expression and deposition of γ-zein-DsRed and by extrapolating this
knowledge to develop strategies that can be applied to pharmaceutical proteins in
commercial processes.
Figure I.12 Schematic overview of the PhD project.
Construction of Model
Fusion Protein (Recombinant DNA techniques)
Generation of Stable
Transgenic plants
(Tissue culture, particle
bombardment, regeneration)
Characterization of
Transgenic Wheat Plants (Fluorescent microscopy, PCR, PAT
analysis)
Protein Analysis
(Isolation of total soluble proteins,
SDS-PAGE analysis, commassie-blue
staining, western blot analysis)
Immunolocalization Studies
(Fixation and embedding, ultra thin
sectioning, fixation for
immunochemistry, fixation for ultra
structure, light microscopy, fluorescent
microscopy and confocal microscopy)
24
II. MATERIALS AND METHODS
II.1. Materials
II.1.1. Chemicals and consumables
All the chemicals used through-out the research work were purchased from the following
companies: Alfa Aesar (Karlsruhe, D), Amersham Bioscience (Freiburg, D), Bio-Rad
(München, D), Boehringer Mannheim (Mannheim, D), Duchefa (Haarlem, NL), Fluka
(Neu-Ulm, D), Gibco BRL Eggenstein, D), Invitrogen (Karlsruhe, D), London Resin
Company Ltd (London, UK), Merck (Darmstadt, D), Molecular Probes (Leiden, NL),
MWG (Ebersberg, D), New England Biolabs (Frankfurt, D), Plano (Wetzlar, D), Roche
(Mannheim, D), Roth (Karlsruhe, D), Serva (Heidelberg, D) and Sigma-Aldrich
(Taufkirchen, D).
The consumables were purchased from: Eppendorf (Hamburg, D), SPI supplies (Adi
Hassel, München, D), Bio-Rad (München, D), MilliPore (Schwalbach, D), Qiagen
(Hilden, D), and Whatman (Bruchsal, D).
II.1.2. Buffers, media and solutions
All the required buffers, media and solutions used in this study were prepared according to
standard protocols as described in Sambrook and Russell (2000). The other optimized
media and solutions are listed here. Heat-sensitive additives such as antibiotic were
prepared in stocks and were filter sterilised by passing through 0.2μm filter (Millipore)
and added to the autoclaved media or buffer after they were cooled down to 55-60°C. The
pH was adjusted with 1M, 5M and 10M NaOH, 1M and 5M KOH or 37 % (v/v) and 10 %
(v/v) HCl.
AP buffer 100 mM Tris-HCl pH 8.9
100 mM NaCl
5 mM MgCl2 , pH 9.6
Coomassie staining solution 0.25 % (w/v) Coomassie blue G-250
50 % (v/v) Methanol
9 % (v/v) Glacial acetic acid
25
Blotting buffer 25 mM Tris-HCl pH 8.3
192 mM Glycine
20 % (v/v) Methanol
Coomassie de-staining solution 20 % (v/v) Methanol
10% (v/v) Glacial acetic acid
DNA extraction buffer 3 % (w/v) CTAB
1,4 M NaCl
0,2 % (v/v) β-Mercaptoethanol
20 mM EDTA, pH 8.0
100 mM Tris-HCl, pH 8.0
1 % (w/v) PVP40
10x DNA loading buffer 0.1 % (w/v) Bromphenolblue
0.1 % (w/v) Xylencyanol
in TBE buffer
KPi buffer for protein extration 1 M KH2PO4 , pH 7.6
1 M K2HPO4
Phosphate buffer (0.1 M) 19 % (v/v) of 200 mM NaH2PO4
81 % (v/v) 9.9 mM Na2HPO4
Protein denaturation buffer 1.05ml KPi buffer
6 % (v/v) EOTH
110 mM (w/v) Urea
1ml (v/v) Methanol
10x PBS 1.37 M NaCl
27 mM KCl
81 mM Na2HPO4
15 mM KH2PO4, pH 7.4
26
Phosphate buffered saline-Tween (PBST) 1x PBS (pH 7.4)
0.05 % (w/v) Tween 20
5x Reducing protein loading buffer 62.5 mM Tris-HCl (pH 6.8)
30 % (v/v) Glycerol
4 % (w/v) SDS
0.05 % (w/v) Bromophenol Blue
10 % (v/v) ß-Mercaptoethanol
5x SDS-PAGE running buffer 125 mM Tris-HCl pH 8,3
960 mM Glycine
0.5 % (w/v) SDS
10x TBE electrophoresis buffer 900 mM Tris-base
900 mM Boric acid
25 mM EDTA, pH 8,3
4X Separation buffer: 1.5 M Tris base
0.4% (w/v) SDS
adjusted pH to 8.8 with HCl
dH2O to 500ml, filter sterilize.
4X Stacking buffer: 30.25g Tris base
2g SDS
adjusted pH to 6.8 with HCl
dH2 O to 500ml, filter sterilize.
2X Sample buffer: 125mM Tris 6.8
6% (w/v) SDS
20% (w/v) glycerol
0.025% (v/v) bromophenol blue.
Fixation solution 4% (w/v) Paraformaldehyde (PFA)
0.2% (w/v) Glutaraldehyde
27
Media and Additives
Escherichia coli
LB (pH 7.0) 1.0 % (w/v) NaCl
1.0 % (w/v) Trypton
0.5 % (w/v) Yeast extract
Glycerol Stock Medium (GSM) 50 % (v/v) Glycerol
100 mM MgSO4
25 mM Tris (pH 7.4)
Antibiotics
Antibiotics Final concentration Stock solution
Escherichia coli
Ampicillin (Amp) 100 mg l-1
100 mg ml-1
H2O
Kanamycin (Kan) 25 mg l-1
100 mg ml- H2O
Agrobacterium tumefaciens
Rifampicin (Rif) 100 mg l-1
100 mg ml-DMSO
Kanamycin (Kan) 25 mg l-1
100 mg ml-H2O
Carbenicillin (Carb) 100 mg l-1
100 mg ml-EtOH 50%
Plants
Phosphinotricin (PPT) 2-3 mg l-1
10 mg ml-H2O
Enzymes and reaction kits
Restriction and ligation enzymes used for DNA digestion were purchased from New
England Biolabs and Fermentas. DNA Taq polymerase produced by the Fraunhofer IME
(Aachen) was used for PCR amplification. The following kits were used:
QIAprep spin Miniprep kit Qiagen
QIAquick® gel extraction kit Qiagen
QIAquick® PCR purification kit Qiagen
QIAquick® plasmid Midi kit Qiagen
28
QIAquick® plasmid Maxi kit Qiagen
Bacterial strains
Escherichia coli strain DH5α (F- (f80d Lac2ΔM15) Δ(LacZYA-argF) U169end A1 rec1
hsdR17(rk- mk-) deoR thi-1 supE44 gyrA96 relA1 λ-, Ausubel et al., 1994) was used as
host cells for plasmid amplification.
II.1.3. Plants
The wheat (Tricitum aestivum) variety BobWhite was used for the generation of stable
transformed plants.
II.1.4. Vectors
The pTRA (Thomas Rademacher, Institut für Biologie VII, RWTH Aachen, Germany)
vector was used for wheat transformation.
II.1.5. Resin and membranes
The Resin from London Resin Company Ltd, UK was used for immunocytochemistry
analysis. HybondTM-C nitrocellulose membranes of size 0.45µm from Amersham
Bioscience and Whatman paper no.1 from Whatman were used in immonoblot analysis.
II.1.6. Equipment
1. DNA gel electrophoresis apparatus: DNA agarose electrophoresis (BioRad, München)
and power supplies (BioRad)
2. Electroporation apparatus: Gene pulserTM, Pulse controller unit, Extender unit
(BioRad) and 0.2 cm or 0.4 cm cuvettes (BioRad)
3. Centrifuges: Eppendorf 5424, 5415R, PICO 21 (Thermo Scientific)
4. PCR Thermocyclers: MJ Mini (BioRad)
5. Photometers: Infinite M200 (TECAN)
6. Sonicator: Transsonic T310 (CAMLAB)
7. Protein gel electrophoresis equipment: Mini PROTEAN supplied from BioRad
8. UV-Transilluminators: Molecular Imager. Gel Doc XR+ imaging system (BioRad)
9. Particle bombardment device: PDS/He1000 particle bombardment system ( BioRad)
29
10. Biobench. SAFE 2010, HERA Guard (Thermo Scientific)
11. Shaker: Innova 4430 (New Brunswick Scientific GmbH, Nürtingen)
12. Microwave: Severin
13. Incubator: MAXQ 8000, Heraus Incubator (Thermo Scientific)
14. Balance: VICON (Acculab), Sartorius (INULA)
15. Vortex: Vortex-2 Genie (Scientific Industries)
16. Oven: Heraeus (Thermo Scientific)
17. Ice Machine: ZIEGRA GmbH
18. pH meter: Hi 221 (Hanna), Phonomenal (VWR)
19. Vibrator: Retsch MM400
20. Light Chamber: RUMED (Rubarth Apparatus GmbH)
II.2. Methods
All the experiments in this study were performed performed in accordance with S1 safety
regulations and were approved by the Regierungspräsidium des Landes NRW. [(AZ 521-
K-1-8/98: AI3-04/1/0866/88 (S1) and 55.8867/-4/93 (greenhouses)].
II.2.1. Transformation, selection and characterisation of recombinant bacteria
Escherichia coli (E.coli) cells were used for plasmid DNA (II.3.1) magnification. These
cells were grown at 37°C either in LB medium (with amp) in a shaker incubator at 225
rpm, or on agar-solidified 1.5% (w/v) LB plates (amp). For glycerol stocks, 600µL of
overnight (o/n) grown liquid culture was mixed with an equal volume of 40% (v/v)
glycerol and placed at -80C° for long term storage.
II.2.2. Preparation of competent E. coli cells for heat-shock transformation
Competent Bacterial (E.coli) strain DH5α cells were prepared for heat shock
transformation. The growth of E.coli was performed with a single bacterial colony
inoculated in 5 ml of LB broth and cultured at 37°C over night (o/n). 500 micro-litres (µl)
30
of fresh o/n culture were added into 100 ml of LB broth and cultured at 37°C for 3-4
hours. When the culture reached an OD600nm of 0.6-0.7 were then transferred to an ice-
cold tube. The cell liquid was placed on ice for 10 min and the cells were recovered by
centrifugation (2,000g/4°C/20 min). The centrifugation step was repeated again. The
pellets were gently resuspended in 200 ml ice-cold sterile distilled water. Then, added to
200 ml 10% (v/v) ice-cold glycerol and finally in 1 ml of 10% (v/v) ice-cold glycerol.
Aliquotes of 200µl of competent cells were frozen immediately in liquid nitrogen and
stored at -80°C.
II.2.3. Transformation of E. coli by heat-shock
The Competent E.coli (II.2.2) was thawed on ice and 10µg of plasmid DNA or ligation
products were mixed gently. The competent cells were put on ice for 30 min. The cells
were incubated for 2 min at 42°C and placed on ice for 2min. After quick cooling 800 µl
of LB media was added to the eppendorf tubes and transformed cells were incubated at
37°C for 1 hour. The volume of 200, 100 and 50µl of cells were plated onto separate LB-
agar plate supplemented with appropriate antibiotics and were incubated at 37°C overnight
until the colonies appeared. Colonies were inoculated for DNA mini and medi
preparations.
II.2.4. Preparation of competent E. coli cells for electroporation
The amount of 5ml of LB medium was inoculated with a single colony of transformed
E.coli strain with vigorous agitation overnight at 37°C. The 5-ml culture was used to
inoculate 500 ml of LB medium at 37°C until the culture reached an OD600nm of 0.6-0.7.
The culture was then removed from shaker and placed on ice for 20-25 min. The bacterial
cells were centrifuged at 3,000g at 4°C for 10 min. The E.coli cells were washed three
times with sterilized water and suspended in ice-cold 10% (v/v) glycerol and 200 µl of ice-
cold distilled water. Finally 40 µl aliquots were stored at -80°C.
II.2.5. Transformation of E. coli by electroporation
The plasmid DNA (II.2.3; 50-100ng) was used for transformation of electro-competent
cells (II.2.1.3). The cells were thawed on ice and the mixture was transferred to the bottom
31
of prechilled (0.2cm) cuvette and assembled into a safety chamber. An electric pulse (25
μF, 2.5 kV, 200 Ω) was applied and then cells were diluted in 1 ml of LB medium and
incubated at 37°C with shaking for 1 h for regeneration of cells. Different volumes i.e 50-
100 µl of the transformed cells were plated onto LB agar (amp) and incubated at 37°C o/n.
II.3. Recombinant DNA techniques
II.3.1. Isolation of plasmid DNA
Plasmid DNA was purified using QIAprep® Plasmid Isolation Mini/medi Kit according to
the manufacturers‟ instructions. Quality and quantity of DNA was confirmed by analytical
agarose gel electrophoresis. Isolated DNA (II.3.1) samples were stored at –20 °C.
II.3.2. Restriction enzyme digestion
DNA restriction analysis was carried out for plasmid DNA (II.3.1) obtained from mini or
maxi-preparation. Restriction buffers were provided by the company and were chosen
according to manufacturer‟s recommendations. Double digests with two restriction
enzymes were carried out in buffer providing 70%-100% activity for either enzyme used.
1ng of plasmid DNA was restricted with 2U of enzyme for 1hrs at 37º C. If restriction
fragments were used for ligation, 0.5-1 µg of vector DNA (II.3.1) and 3-8µg of insert
DNA depending on the ratio of the insert with the plasmid were digested with 10U of
restriction enzyme for 2hrs are recommended temperature. Reactions were stopped by
freezing at 20°C until further processing. Fragments were subsequently separated by
agarose gel electrophoresis and photo-documented.
II.3.3. Agarose gel electrophoresis
Undigested plasmid DNA (II.3.2), PCR fragments (II.3.6) and enzyme restricted plasmid
DNA (II.3.2) was electrophoretically separated with agarose gels. Analysis for Plasmid
DNA from mini/midi preparation or digestion (II.3.2) or ligation fragments (II.3.4) were
separated with gel electrophoresis as described (Frisch et al., 1996) in 1.2% (w/v) agarose
gels supplemented with ethidium bromide (0.1g/ml). Ethidium bromide as visual marker
was added to the gel solution and TBE electrophoreses running buffer before to the
experiment. Known amount of DNA marker such as 1Kb and 100bp ladder digested with
32
PstI were used as size and concentration control. The DNA bands were visualised directly
upon illumination with a UV transilluminator at 302 nm. The pictures were taken with
camera and documented.
II.3.4. Ligation of DNA
The digested DNA fragments (II.3.2) were ligated 1μl Quick T4 DNA-Ligase (NEB) in
buffer systems recommended in the manufacturer‟s protocol in a final volume of 20μl.
The vector–insert ratio was calculated depending on the size of insert in relation to total
plasmid size. The ligation was carried out for 16 hours at 4º C. Sticky-end ligations were
carried out at room temperature for 5 to 30min depending on the ligase used.
II.3.5. DNA isolation
Total genomic DNA from wheat was isolated from young wheat leaves. Approximately
10cm long, young and fresh leaves were collected from plants and immediately frozen into
liquid nitrogen and were homogenized with pestle. Five hundred microlitre DNA
extraction buffer (1% (w/v) SDS, 100mM NaCl, 100mM Tris base, 100mM Na2EDTA,
pH=8.5 by HCl) was added to the crushed wheat leaf material. Equal volume (500l) of
phenol: chloroform: isoamylalcohol (in a ratio of 25:24:1) was then added and tubes were
shaken vigorously until a homogenous mixture was obtained. Samples were then
centrifuged at 5000rpm for 5 minutes. The aqueous phase was transferred to a fresh 15ml
tube. One-tenth volume (50l) of 3M sodium acetate (PH= 4.8) and equal volume (500l)
isopropanol was added in the tube to precipitate the DNA. The samples were then
centrifuged at 5000 rpm for 5min to make the DNA pellet. After removing the
supernatant, the pellet was washed with 70% (w/v) ethyl alcohol and was dried at room
temperature for an hour and re-suspended in 40l TE buffer (10mM Tris, 1mM EDTA and
PH = 8.0). To remove RNA, DNA was treated with 40g RNAse-A at 37C for 1 hour.
After RNAse treatment, DNA samples were stored at 4C. For PCR analysis, the wheat
DNA was diluted to 1: 4 in highly purified water.
33
II.3.6. Polymerase chain reaction
The Polymerase Chain Reactions were carried out using modified protocols of Devos and
Gale (1992). For PCR, DNA primers (Table II.1) weredeveloped for DsRed sequences.
Total of 25l PCR reaction was used containing 50-100ng total genomic DNA template,
0.25M of each primer, 200M of each dATP, dGTP, dCTP, dTTP, 50mM KCl, 10mM
Tris, 1.5mM MgCl2 and 2.5 units of Taq DNA polymerase. The PCR conditions were set
for amplification of the DNA. i) Denaturation step of 4 minute at 94C ii) 40 cycles each
consisting of a denaturation step of 1 min at 94C iii) annealing step of 1 minute at 34C
and iv) an extension step of 2 minutes at 72C. The last cycle was followed by 10 minutes
extension at 72C. All amplification reactions were performed using (name of the PCR in
the lab) programmable thermocycler. The amplification products were electrophoresed on
1.5 % (w/v) agarose/TBE gel, and visualized by staining with ethidium bromide (EtBr)
under ultra-violet (U.V) light. Sequence information for primers are given in Table II.1.
Table II.1: The sequences information of the primers used for DsRed detection.
II.3.7. Purification of DNA from agarose gel
Gel electrophoresis (II.3.3) was used to purify the desired DNA fragments (II.3.7)
obtained from restriction/ligation (II.3.2; II.3.4) of plasmid DNA (II.3.1). The fragments
of interest were made visible under UV light and cut out of the gel using a new razor blade
for each band. DNA was purified using QiaQuick spin columns (Qiagen, Studio City, CA)
following the manufacturer‟s protocol. Total elution volume was 50µl for further analysis.
S.No
Oligo Name Sequence (5'-3') Size (bp) Applification
product(bp)
1 DsRed 56e TACCCTGGTAGCTGCACAGG 20
362
2 DsRed 55e TATGTCAAGCACCCTGCCGA 20
34
II.3.8. Determination of the DNA concentration
The purified DNA was analysed by spectrophotometry by measuring the absorbance of the
solution at 260 nm, the wavelength at which both DNA and RNA absorb maximally to
determine the DNA concentration, 50µg/ml DNA equal an absorbance of 1 at 260 nm
(Fritsch et al., 1996).
II.3.9. DNA sequencing
A single plasmid colony (II.3.4) was amplified with by Qiagen minipreparation or
QiaFilter maxi preparation (II.3.1). The correct nucleotide sequencing of expression
construct was verified by sequencing of the purified DNA. The nucleotide sequencing was
performed at Fraunhofer Institute for Molecular Biology and Applied Ecology (IME),
RWTH Aachen using an ABI DNA sequencing apparatus for evaluation of sequence
correctness; results were compared with published sequences using Clone manager
software. The clones containing correct sequences were used in further studies.
II.4. Wheat transformation
Immature wheat (Triticum aestivum L. cv Bobwhite) were used as explants and grown in
the light chambers at Institute for Molecular Biotechnology (Biology-VII), RWTH
Aachen University and at Vienna Institute for Biotechnology, Department of Applied
Genetics and Cell Biology (DAGZ), University of Natural Resources and Life Sciences
(BOKU), Vienna, Austria. Plants were germinated in soil at 14ºC under 16h photo-period
light for three week until plantlets produce 3-4 tillers per plants (about three weeks). The
plantlets were taken to 25ºC day/night, 16h light. Pests and diseases were kept to
minimum by using cold treatment of soil at -80ºC for 2 days and by good housekeeping
practices. Plants with disease symptoms were discarded immediately. Immature embryos
of 13-15 days after anthesis (DAA) were used as starting material for wheat
transformation.
35
II.4.1. Isolation and pre-culture of immature embryo
The wheat seeds of 13-15 DAA were selected for isolation of immature wheat embryos.
All procedures were done microscopically in a sterile environment. The immature
embryos were removed with a size of approximately 0.5-1.5 mm which are generally most
responsive for callus induction. The germination response is relatively higher with such
embryos but there is genotypic variation as well. For each 9 cm petri-dish approximately
20-30 scutella were placed on induction medium (Table II.2). The embryos were located
within a central target area and orientating them with the uncut scutellum uppermost i.e.
the uncut side is bombarded (see Fig. III.5A). As negative control, 20 embryos were
placed on induction media for bombardment only with gold particles and without DNA.
Prior to bombardment, the donor material was incubated in the dark at 26 ºC for the next
5-6 days.
II.4.2. Preparation of DNA coated gold particles
The preparation of gold particle and coating was performed according to Sanford et al.,
1993. Prior to DNA coating, gold particle stocks of 150 ml containing 150 mg of gold
particle (0.6 – 0.9 µm) in size were prepared. For bombardment, 25mg of gold particles
were thawed and ultra-sonicate for 15sec. Gold particle were coated with plasmid DNA
with concentration of 20µg at final volume of 1 ml. The required amount of DNA was
adjusted, depending on the concentration of DNA. The required amount of DNA was
mixed with 25 mg of gold particle and double distilled water up to 100µl with constant
vortex. Added 200µl CaCl2 (2.5 M) and continued vortexing for 2 min. 100µl of fresh
made spermidin (0.1 M) was added to the mixture and vortex for additional 10 min. The
supernatant was removed after centrifugation at 13,000 rpm for 5 min. 500 ml of ice
chilled 100% ethanol was added and sonicated briefly. The washing was repeating again
with centrifuge at the same condition and supernatant were removed. 1ml of 100% ethanol
from the freezer were added to the pellet and sonicated for 15 sec. 160 µl of coated
solution was used for loading the macro-carriers.
36
II.4.3. Micro-projectile bombardment
The PDS-1000/He particle gun (BioRad, Munich, Germany) delivery system was used for
the delivery of coated DNA into 5-6 days old wheat embryos. For using biolistics,
appropriate safety precautions described by the manufacturer were followed.
All the accessories required such as rupture disks (650, 900 and 1100psi), micro and
macrocariers, rupture disc holder, stopping screen, and gold particles (0.6-0.9 µm) were
purchased from BioRad. A tank containing Hellium (He) gas with purity grad 5.0 was
used for pressure. A pump was used to create a desired amount of vacuum inside the
bombardment chamber of the gene gun.
Recommended settings were followed for PDS 1000/He as optimized: gap 2.5cm,
stopping plate aperture 0.8 cm, target distance 5.5cm, vacuum 28 Hg, vacuum flow rate
5.0, vent flow rate 4.5. All the equipments were sterilised with 70% (v/v) ethanol.
Sterilised the macro-carrier holders, macrocarriers, stopping screens and rupture discs by
dipping in 100% ethanol and allowed to evaporate completely before use. The DNA
coated gold particles were briefly vortexed before 160 ul of coated gold were taken.
Placed centrally onto the macro-carrier membrane and allowed to dry. Rupture disc of 650
psi and 950 psi was placed into rupture disc retaining cap and tightened. Macro-carrier
holder containing macro-carrier gold particles/DNA was placed over the stopping screen
in the nest that maintained its position using the retaining ring. Placed a sample on the
target stage of 2.5 cm gap, draw a vacuum of 27-28 Hg and fired the gun.
II.4.4. Tissue culture and selection of transformants
Immature wheat embryos (IEs) were cultured on MS-based (Murashige and Skoog, 1962)
Callus Induction Medium (W-ID), Induction Selection Medium (W-SM), Regeneration
Medium (W-Z10), Osmotic Medium (W-Osm) and Rooting Medium (W-R), with macro
elements, micro elements, recommended additives and plant hormones. The media were
solidified with 0.2% (w/v) gelrite. The ingredients for each media and other supplements
are listed in Table II.2.
37
Immature wheat zygotic embryos were used as explant for transformation. IEs were
isolated aseptically and bombarded (II.4.3) with DsRedzenH construct. Isolated IEs were
cultured immediately on W-ID medium and kept under dark at 26°C for 5-6 days. The IEs
were transferred to W-Osm 4 hours before bombardment and were transferred to W-SM
after 16-20 hours after bombardment. The Callus induction medium contained 2mg/L 2, 4-
D as auxin source for initiation and proliferation of calli while MS was used as nutrient
source for plant material in all tissue culture media. For selection of transformed cells the
proliferating calli were started one week after bombardment by transferring to W-SM
containing 2mg/L of herbicide Phosphenothricin (PPT) where selection continued for
transgenic Calli on SM-2 for two weeks in the dark at room temperature. Embryogenic
calluses are subsequently transferred to regeneration media (W-Z10) for shoot induction
under light at standard tissue culture conditions with 26°C temperature and 2000 lux light
intensity for 16h per day. Wheat regenerating media was supplied with zeatin as growth
hormone with MS and 3mg/L of PPT. Shoots regenerated from transgenic calli were
observed within three weeks on regenerating media (W-Z10). The calli were sub-cultured
on to fresh, hormone-free medium every three weeks. Shoots and plantlets of 3-5cm size
were transferred to ½ strength MS with 3mg/L PPT selection for proper plant
development. After three to four weeks plantlets of reasonable size and with developed
root system and were transferred to growth rooms in normal soil.
38
TableII.2: Media ingredients used for wheat tissue culture.
Ingredients
W-ID
W-SM
W- Osmoticum
W-Z10
W-RM
MS Basal Salts/g 4.39 4.39 4.39 4.39 2.19
Sucrose/g - - 20 10
Manitol/g - - 36.4 - -
Maltose/g 40 40 30
Sorbitol/g - 36.4 - -
Glutamine/mg 500 500 500 - -
2,4-D/mg 2 2 2 - -
Zeatin/mg - - - - -
PH w/ NaOH 5.8 5.8 5.8 5.8 5.8
Gelrite/g 2.5 2.5 2.5 2.5 2.5
After autoclaving
PPT/mg - 2 - 2 3
Zeatin - - - 10
Vit-mix (500X)/ml 2 2 2 2 1
Temperature (°C) 24-25 24-25 24-25 24-25 24-25
Stock Ingredients
MS Vitamins
Using Duchefa stock powder, 1000X solution were prepared and filter sterilised with a
0.25um filter. Stored at 4°C for maximum duration of 3 months.
Stock solutions
Make up stock of 4mg/ml 2,4-D in 100% (v/v) Ethanol and were stored at 4°C. Prepared
stock of 10mg/ml of PPT in 70% (v/v) ethanol. The solutions were stored indefinitely at –
20°C.
39
Table II.3: List of ingredient and suppliers
Ingredient Supplier Catalogue Number
MS Basal Salts Duchefa M0221
MS Vitamin Stock Duchefa M0409
Sucrose Grade I Sigma S5390
Sucrose Grade II Sigma S5391
Mannitol Roth NA*
Sorbitol Roth NA*
L-Glutamine Sigma G8540
Casein Hydrosylate Sigma C7290
2,4-D Duchefa D0911
Zeatin Sigma (also Duchefa) Z0164
*NA. Not available.
TableII.4: MS vitamin ingredients
Made up to 500X solution with 100ml distilled water. Filter sterilised with a 0.25um filter
and stored at 4°C for up to 3 months.
Ingredients
Amount (mg)
Myo-Inositol 5000
Glycine 100
Nicotinic Acid 25
Pyridoxine HCl 25
Thiamine HCl 5
40
II.4.5. Selection and propagation of transgenics
Selection of transgenic plants was carried out by three methods listed below.
II.4.5.1. DsRED expression
The cells carrying transgene can be easily screened by DsRed expression under
fluorescent microscope. The red fluorescence can be observed in embryo cells after 18
hours of the bombardment and the images were documented.
II.4.5.2. PPT assay for transgenic plants selection
Transgenic wheat plants were selected by spraying the herbicide solution (PPT) on two
weeks old plants. Solution contained 150mg/L of PPT were applied twice after
transferring the regenerating plantlets to the soil. The plants were treated by spraying or
dipping the wheat leaves into PPT solution. The non-transformed plants were dried or died
with the herbicide application facilitating the final transgenic selection. Wheat plants
surviving under selection pressure were grown in growth rooms under standard conditions
till maturity and the seeds were harvested to advance to the next generation.
II.4.5.3. Regeneration and segregation on selection media
T1 generation wheat seeds were soaked overnight in distilled water and the embryos were
removed aseptically and cultured on MS half media with 3.0 mg/L of PPT. The transgenic
lines showed fast regeneration compared to wild type (Wt) BobWhite wheat embryos as
negative control. The transgenic lines showed a 3:1 segregation pattern. A total of 50
embryos were checked for regeneration and segregation. The wheat line resistant to
herbicide phosphenothricin (PPT) was used as positive control.
II.5. Protein analysis
II.5.1. Isolation of total soluble proteins from wheat tissues
Total proteins were extracted from wheat leaves, endosperm and embryo of the transgenic
lines (II.5.1). Using vibrator, the three tissues were frozen in liquid nitrogen and grounded
to a fine powder. The samples were homogenized with 3 volume of protein extraction
41
buffer (II.1.2). Centrifuge for 15sec and the 100ul of supernatant was added to new 1.5
eppendorf tube. Centrifuge again for 40 min at 13,000 rpm at 4°C. Discarded the
supernatant and the pellet was re-suspended in 5X loading buffer for further analysis.
II.5.2. SDS-polyacrylamide gel electrophoresis of proteins
SDS-polyacrylamide gels (SDS-PAGE) were used for separation of proteins with stacking
and separating gels (II.1.2). The plant extract (II.5.1) re-suspended in 5x protein loading
buffer, were analysed using 12% Polyacrylamide (PAA) gels following manufacturer‟s
instructions. Samples were denatured by incubation in a boiling water-bath or on heater at
100°C for 10 min. SDS-PAGE was performed using BioRad electrophoresis apparatus
with timely increasing voltage of 50V for 5min, 100V for 10min, and 150V for 10min and
finally at 200V for 90 min or until the bromophenol blue front ran out of the gel.
II.5.3. Coomassie brilliant blue staining
The proteins separated on SDS-PAA gels were revealed by staining with Coomassie
Brilliant Blue for 1 hour in Coomasie staining solution (II.1.2) at RT under constant
rocking. Coomassie staining was removed with de-staining solution (II.1.2) until the bands
were clearly visible. SDS-PAA separated proteins were analysed to Western blot (II.5.4).
The pictures were saved for documentation.
II.5.4. Western blot
Electrophoretically separated protein by SDS-PAA gels (II.5.2) were analysed for gamma-
zein with specific antibodies. In the presence of transfer buffer, Electrotransfer was carried
out with the BioRad Mini-Transfer apparatus to HybondTM-C nitrocellulose membrane
(0.45um). The transfer was done under constant cooling at 60V for 90 minutes. The
membrane was washed 3 times with PBS-T and once with PBS. The successfully
transferred protein membrane was blocked with PBS buffer (II.1.2) containing 5% (w/v)
skim milk for one hour. The membrane was washed 3X with PBS-T and once with PBS.
Then, the membrane was labelled with the primary antibody (Rabbit 27kD γ-zein) at
dilution of 1/1000 for overnight at 4 ºC. The protein membrane was kept 30min at room
temperature, followed by 3x washing with PBS-T and once with PBS. The the bound
42
antibody was revealed by addition of secondary antibody (Anti Rabbit IgG) for 1hours at
concentration of 1/5000 at room temperature. The membrane was washed three times with
PBS-T and once with PBS buffer. The targeted protein was finally detected by substrate
NBT/ BCIP (BioRad) in distilled water for 15min or until the bands appeared.
II.6. Immunolocalization studies
II.6.1. Fixation and embedding
Different wheat tissues i.e. leaf, endosperm and embryo were fixed for
immunocytochemistry and structural analysis. Wheat samples were processed for
immunolocalization and ultrastructural analysis as described previously by Arcalis et al.,
2004.
II.6.2. Fixation for immunocytochemistry
The samples were sliced mm³ and fixed in fixed in 4% (w/v) paraformaldehyde and 0.2%
(v/v) glutaraldehyde in phosphate buffer (II.1.2). Samples were incubated in fixation
solution for overnight at 4°C. The samples were washed 5 times each with 0.1M for
15min. Dehydration was carried out first by passing the specimens through an ethanol
series (Table II.6). Samples were incubate each for 90 min in 50% (v/v), 70% (v/v) at 4°C
and followed by 96% (v/v) and 100% (v/v) ethanol at room temperature, each for the same
time. Specimens were therefore infiltrated in LR White Resin (London Resin Company
Ltd, UK), in a series 1:1, 1:2, 1:3 ethanol:resin (v/v), for 1 h each, and 100% (v/v) resin
overnight at 4°C. The specimens were transferred to inclusion molds filled with pure resin
avoiding air bubbles. The samples were allowed to polymerize at 60°C for 48 h. Then,
100-nm ultrathin sections (for electron microscopy) or 1μm sections (for light
microscopy) were obtained using a Leica Ultracut E ultramicrotome and collected on
Formvar-coated 200 mesh gold grids (Sigma) for electron microscopy or on glass slide for
fluorescent microscopy.
II.6.3. Fixation for ultra-structure
Wheat samples including endosperm, leaves and embryos tissues were sliced in 1mm size
and fixed overnight in fixation solution (II.1.2) at 4°C. The vibratome sections were
43
washed 5 times for 15min with 0.1M phosphate buffer (PB). The samples were processed
in 1% (w/v) osmium in 0.1M phosphate buffer (PB) supplemented with 0.8% (w/v)
KFeCN for 3 hours. The osmification was performed under fume hood and used osmium
solution was discarded in a container filled with plant oil. Samples were washed with
0.1M phosphate buffer (PB) 5 times for 15min each. Dehydration was followed after
washing in a series of acetone solution. The acetone dehydration series was carried out
according to the table (Table II.5)
Table II.5. Series of acetone dehydration used for ultra structure fixation.
The specimens were infiltrated in different mixture of ethanol and spur resin as given in
the table below. The specimens were heated to polymerize at 60°C for 48 h.
Table II.6. Mixture of resin/ethanol used for ultra structure fixation of the tissues.
Resin/Ethanol (v/v) Time Temperature
1:3 3 Hours RT
2:2 3 Hours RT
3:1 3 Hours RT
Pure resin Overnight 4 °C
Acetone % Time (min) Temperature (°C)
30 1*10 4
50 2*10 4
70 3*10 4
96 3*10 4
100 3*15 4
44
The ultra-thin sections were made using Leica Ultracut E ultramicrotome. 100-nm
ultrathin sections were used for electron microscopy and 1-μm sections for light
microscopy and fluorescent microscopy.
II.6.4. Light and electron microscopy
Ultrathin sections were cut using a Leica Microsystems Ultracut UCT, mounted on 300-
mesh nickel grids and immunogold labeled. Grids were floated on drops 5% (w/v) bovine
serum albumin (BSA) in 0.1M phosphate buffer for 15 min at room temperature. The
sections were incubated with primary antibody (Polyclonal DsRed) for overnight at 4°C.
The primary antibody was diluted at 1:500 as prescribed by the manufacturer. The sections
were washed with PBS-T two times for 10 min at room temperature. Incubated with
secondary antibody i.e. Goat anti-Rabbit Alexa-546 at the concentration of 1:30
conjugated with 15nm gold particles. At room temperature, washed twice with phosphate
buffer (0.1M) for 10min. Washed again two times for 10min each. The samples were air
dried before used for electron microscopy analysis. Controls were processed following the
same protocol. For light microscopy, the samples were stained in toluidine blue and
starches were stained by incubating section with Lugol‟s iodine solution for 5min.
II.6.5. Immunofluorescent labeling
The wheat seed and leaf samples were fixed as described (Arcalis et al., 2004). The 60 to
100 µm vibratome sections were prepared and dried well. The specimens were blocked
with bovine serum albumin (BSA) in 0.1M phosphate buffer pH 7.4 for 15min at room
temperature. The specimens were ncubated with primary antibody (DsRed Polyclonal
antibody) at concentration of 1/500 overnight at 4°C. The thin sections were washed with
PB-T (II.1.2), followed by incubation with secondary antibody (goat anti Rabbit, Alexa
546) at dilution of 1/30 in 0.1M phosphate buffer under dark conditions. The samples
were washed with phosphate buffer (0.1M, pH 7.4). Finally the thin sections were washed
with distilled water and air dried in dark for confocal and florescent microscopic analysis.
45
II.6.6. Fluorescence microscopy
Fluorescent markers (DsRed) exclude the sample preparation for protein detection in plant
cells. Therefore the wheat seed and leaf samples were cut in 0.1 M phosphate buffer pH
7.4 and applied directly for fluorescent microscopy analysis. Red fluorescent images were
detected under the green light filter.
II.6.7. Confocal microscopy
The wheat seed and leaf containing DsRed as fluorescent marker did not require any
sample preparation for confocal microscopy. Therefore, the samples were cut in 0.1 M
phosphate buffer pH 7.4 and applied examined for confocal microscopy analysis. Red
fluorescent images were collected after 543 nm excitation using a 550-600 nm emission
window.
46
III. RESULTS
The maize seed storage protein γ-zein is a class of prolamin that can induce the formation
of protein bodies in its native environment, the endosperm. However, the expression of γ-
zein in diverse eukaryotic cells has shown that it also induces the formation of novel
compartments in many heterologous backgrounds making it potentially useful as a more
general approach for recombinant protein expression. We generated an expression
construct comprising 90 amino acids sequences i.e repetitive (VHLPPP)8 and Pro-X
domain of maize prolamin (γ-zein) fused with Discosoma spp. red fluorescent protein
DsRed under the control of a constitutive ubiquitin promoter (Fig. III.1), allowing the
protein to be expressed in both seed and non-seed tissues in wheat plants. Transgenic
wheat lines produced by particle bombardment (II.4.3) were used to analyze the
expression and distribution of the recombinant model proteins, focusing on the formation
of fluorescent protein bodies which were investigated using a range of molecular,
immunological and imaging methods. Recombinant fusion protein (γ-zeinDsRed)
accumulated in the artificial protein bodies in all tissues analyzed. The subcellular
localization of constitutively expressed recombinant fusion proteins were examined in
both seed and green tissues. The possible degradation of the fusion proteins was
investigated by immunoblot analysis (II.5.4) and their accumulation within novel protein
bodies was confirmed by the immunofluorescence labeling (II.6.5) of leaf, endosperm and
embryo tissues from transgenic wheat plants.
III.1. Fusion protein expression construct
The fusion protein containing the entire DsRed coding sequence joined to the 90 amino
acids of γ-zein comprising proline rich repetitive (PPPVHL)8 and Pro-X domain. The
fusion gene was inserted into the pTRA vector downstream of the constitutive ubiquitin
promoter, and in frame with a His6 tag to facilitate affinity purification. The vector
backbone included an Escherichia coli origin of replication and ampicillin resistance gene
for cloning, and the bar resistance gene conferring resistance to the herbicide
phosphinothricin (PPT) for selection in plants. Transcription bar gene is also driven by the
constitutive promoter ubiquitin. The final expression vector was named DsRedzenH (Fig.
III.1).
47
12386 bps
2000
4000
6000
8000
10000
12000
AscI
SalIXhoI
NcoI
SalIBamHI
SalI
SacIIKpnI
SalIXhoI
NcoIEcoRINcoI
BstXI
NotIXhoISacI
BamHI
PmeI
MscI
PvuI
SapI
SAR
Pubi
5'UT
intron
bar
pA35S
SAR
Pubi 5'UTintron
CHS
LPHglycoDsRed
gamma-zeinhis6-tagpA35S
SAR
RB
RK2 ori
bla
ColE1 ori
LB
Figure III.1 Vector map of pTRAbux-DsRed.zen-H (DsRedzenH). E. coli DH5α competent cells (II.2.2)
were transformed by heat shock (II.2.2) and single colonies were tested by PCR (II.3.6) to confirm the
presence of an insert (II.3.9). The recovered plasmids (II.3.7) were digested with restriction enzymes (II.3.2)
to verify the orientation of the insert, and its integrity was confirmed by sequencing (II.3.9).
48
Figure III.2 Structure of the pTRAbux-DsRed-zenH (DsRedzenH) expression vector.
III.2. Transformation of wheat plants
Approximately 1500 immature wheat embryos were bombarded (II.4.3) with DNA-coated
metal particles (Fig. III.4) and the tissues were cultured for 14 days to induce the
formation of callus. Individual explants were transferred to selection medium (Table II.2)
containing 2 mg/ml PPT and subcultured onto the same medium at regular intervals (Fig.
III.5). Following selection (II.4.5), independent transgenic callus lines were placed on
regeneration medium (Table II.2) containing 3 mg/ml PPT for 3 weeks in natural light.
Transgenic shoots ~15 cm in length were transferred to soil in the greenhouse, and
checked for PPT resistance after 2–3 weeks by spraying with 250 mg/L PPT every 2 days.
Ten transgenic plants were recovered but only three were selected for further analysis
based on highest expression of DsRed in leaves (II.4.3) and were cultivated in the
greenhouse until mature. The wheat transformation protocol is summarized in Figure III.3.
49
Figure III.3 Wheat plant transformation procedure. A. Immature wheat embryos were isolated
aseptically 14 days after anthesis (II.4.1) and maintained on induction medium (Table II.2) for 6 days in
darkness. B. Six-day-old embryos were placed on osmoticum medium (Table II.2) before bombardment C.
Bombardment was carried out using the PDS-1000/HE (II.4.3) D. Fluorescence imaging (II.6.6) confirmed
DsRed expression in wheat cells 24 h post-bombardment. E. Transgenic wheat callus pieces began to
regenerate under illumination. F. Three-week-old wheat plants continued to flourish on regeneration
medium (Table II.2) G. Transgenic plantlets developed on ½MS medium (Table II.2) containing PPT
(II.4.4) H. The plantlets were transferred to the greenhouse and maintained under selective conditions
(II.4.4). T. Transgenic wheat plants were grown to maturity in the greenhouse.
50
Figure III.4 Bio-Rad PDS1000/He particle bombardment apparatus.
Figure III.5 Wheat tissue culture: from immature embryos to shoot development. A. Immature
embryos (14 days after anthesis) on induction medium (Table II.2). B. Seven-day-old wheat embryos pre-
bombardment (II.4.3) C. Wheat callus 5 days post-bombardment (II.4.3) D. Three-week-old transgenic
callus on selection medium (II.4.5.2) E. Wheat callus producing shoots (II.4.5.3) on regeneration medium
(Table II.2) under illumination. F. Shoot development after 3 weeks on regeneration medium (Table II.2)
under illumination.
51
III.3. Selection of wheat plants using PPT
Wheat callus derived from immature embryos was bombarded (II.4.3) with plasmid
DsRedzenH (Fig. III.1) containing the bar gene for selection with PPT (Fig. III.5). When
the primary transformants reached maturity, T1 seeds were harvested to generate T1 plants
for segregation analysis under PPT selection (II.4.5.3). We analyzed 20 T1 plants for each
line (WA-07, WB-21 and WB-28) by applying an aqueous solution containing 150 mg/L
or 250 mg/L PPT on the 15th
and 25th
days after germination (Fig. III.6). Two weeks later,
the appearance of the plants was recorded and the surviving plants were evaluated for the
presence of the transgene. All three transgenic lines produced plants that were resistant to
PPT whereas the sensitivity of all wild-type plants was apparent (Fig. III.6).
The segregation ratio was determined by harvesting 50 immature seeds at least 15 days
post-anthesis from each line, surface sterilizing the seeds and rescuing the embryos
(II.4.1). The embryos were placed on ½MS medium (Table II.2) containing 3.0 mg/L of
PPT and the proportion of resistant and sensitive embryos was determined (Fig. III.7).
Transgenic PPT-resistant wheat plants were used as positive controls and wild-type plants
as negative controls (Fig. III.7). We observed the expected 3:1 Mendelian segregation
ratio for all three transgenic lines (WB-21, WB-28 and WA-07) after 10 days, indicating
single-locus integration.
52
Figure III.6 Wheats plant after PPT application. The transgenic wheat leaves were examined under
fluorescence microscope (II.6) for DsRed expression and were selected for PPT analysis. The plants were
sprayed with an aqueous solution containing 150 mg/L or 250 mg/L PPT on the 15th
and 25th
days after
germination. Two weeks later, the appearance of the plants was recorded and the surviving plants were
evaluated for the presence of the transgene.
A, B, C. Wild type cv. Bobwhite plants are affected with PPT application. D, E, F. Transgenic wheat lines
WB-21, WB-28 and WA-07, respectively, showing resistance to PPT.
53
Figure III.7 Segregation of transgenic wheat lines: Embryos were removed aseptically from the
transgenic wheat seeds and were placed on ½MS medium (Table II.2) containing 3.0 mg/L of PPT. The petri
dishes were placed in growth chamber at 25 ˚C for one week and germination of the embryos was observed.
1. Growth of T1 embryos after one week on ½MS medium (Table II.2) with PPT. A, B. Transgenic line WB-
21. C, D. Transgenic line WB-28. E, F. Transgenic line WA-7 shows high resistance to PPT with normal
germination N. No germination was observed for wild type wheat (cv. Bobwhite) used as negative control 2.
Growth of wheat embryos after 10 days on ½MS medium (Table II.2) to confirm growth of positive and
negative controls on PPT containing media. A Normal growth was observed for positive control (PPT-
resistant wheat line) B. Embryos show no germination for wild type wheat cv. Bobwhite used as negative
control.
54
III.4. Molecular verification of transgenic wheat lines
Transgene integration in the wheat lines was confirmed by testing immature embryos of T1
plants rescued under PPT selection (II.4.6.2) by PCR (II.3.6). Germinating embryos
(Fig.III.5) developed into plants after transfer to soil (Fig.III.3T). The total genomic DNA
was extracted from leaves (II.3.5) and amplified using forward and reverse primers
specific for the DsRed transgene (Table II.1). The anticipated 362-bp product was
identified in all three transgenic lines (Fig. III.8) matching the plasmid positive control (II.
3.1). There was no product amplified in the negative control (wild-type wheat cv.
Bobwhite).
Figure III.8 Confirmation of transgene integration by PCR. PCR was performed as described (II.3.6)
for total genomic DNA isolated (II.3.5) from transgenic wheat leaves. 5µl from 25 µl reaction was applied
on to 1.2% TAE agarose gel (II.3.3). PCR products of 362 bp were amplified using primers designed for
DsRed (Table II.1). All three transgenic wheat lines (WB21, WB 28 and WA07) yielded a 362-bp
amplification product, matching the positive control, DsRedzenH (II.3.1). There was no band in the negative
control lane corresponding to wild-type wheat leaves. 100bp ladder was used as marker.
55
III.5. DsRed as a marker for selection and protein
trafficking
Fluorescent marker DsRed was utilized as fusion protein to monitor the expression of
recombinant proteins in transgenic wheat tissues. As fusion, DsRed was found very
beneficial for transgene selection at very early stages of transformation process.
Fluorescence microscopy (II.6.6) revealed strong DsRed fluorescence in transgenic callus
18 h after bombardment (Fig.III.10). A more detailed time-course analysis confirmed
DsRed fluorescence in rapidly-dividing callus cells (Fig. II.10 A, B, C, D, E, F), allowing
these cells to be selected for regeneration. Transgenic plants from lines WA-7, WB-21 and
WB-28 showed a high and consistent level of transgene expression in the leaves, and were
selected for further analysis (Fig. III.11A, B,C). Leaves from wild type plants showed no
evidence of fluorescence (Fig. III.11D).
DsRed was also used as fluorescence marker for trafficking of recombinant proteins,
reflecting the detection of labeled protein bodies by fluorescence (II.6.6) and confocal
microscopy (II.6.7) from 8-10 days post anthesis. In addition, the fusion protein could also
be detected using DsRed-specific antibodies for immunofluorescence analysis (Fig. III 15,
16 and 18). The γ-zein-DsRed protein was also identified immediately after the isolation
of total soluble protein from transgenic wheat leaves and seeds (III.5.1) when the protein
pellet was illuminated with green light (Fig. III.9). No DsRed fluorescence was detected in
extracts from the wild type cv. Bobwhite (negative control).
56
Figure III.9 Protein pellets extracted from wheat leaves under green light. Leaf tissues from transgenic
wheat line (WA-07) were homogenized with 3 volume of protein extraction buffer (II.1.2). The mixture was
centrifuged for 40 min at 13,000 rpm at 4 ˚C and examined under green light with red filter.
A. Red fluorescence was observed in the bottom pellet shows evidence of recombinant proteins
accumulation in transgenic wheat leaf. B. Protein extracts (II.5.1) from wild type wheat cv. Bobwhite protein
shows no fluorescence.
57
Figure III.10 DsRed expression during transgenic callus development. Wheat immature embryos (after
14 days post anthesis) were placed on W-ID medium (Table II.2) for six days under dark condition at room
temperature. The plasmid DNA (II.3.1) was coated with gold particles (II.4.2) and regenerating wheat calli
were bombarded as described (II.4.3).
Fluorescence images (II.6.6) A, B, C, D, E, F taken 1, 2, 6, 12, 16 and 20 days after bombardment (II.4.3).
The expression of recombinant protein (γ-zein-DsRed) is visible in growing wheat calli 18 hours after
bombardment (II.4.3).
58
Figure III.11. DsRed as a visual selection marker in transgenic wheat lines. Young transgenic leaves
were cut into small pieces in 0.1 M phosphate buffer, pH 7.4 (II.1.2) and examined for the expression of
recombinant proteins.
Fluorescence microscopy images (II.6.65) confirms DsRed transgene expression in A. WB-21, B. WB-28
and C. WA-7 transgenic leaves, compared to D. No red fluorescence was observed in wild-type cv.
Bobwhite used as negative control. Bars, 500 µm (A, B, C, D).
59
Figure III.12 Expression of induced protein bodies in wheat endosperm cells. Transgenic wheat seeds
were analyzed 17 days post anthesis for recombinant protein production in wheat endosperm cells. The seeds
were sliced in 0.1 M phosphate buffer, pH 7.4 (II.1.2) and observed under florescence microscope (II.6.6).
Fluorescence microscopy (II.6.6) images are shown at progressively higher magnifications. Numerous
DsRed bodies were observed throughout the endosperm cells confirmed the accumulation of recombinant
proteins (γ-zein-DsRed) in wheat storage tissues. Bars 500 µm (A), 200 µm (B), 100µm (C), 50µm (D).
60
Figure III 13. Expression of DsRed protein bodies in wheat embryo cells. Transgenic wheat seeds were
analysed 17 days post anthesis for recombinant protein production in wheat embryo cells. The embryos were
isolated and sliced in 0.1 M phosphate buffer, pH 7.4 (II.1.2) and observed under florescence microscope
(II.6.6).
Fluorescence microscopy (II.6.6) images are shown at progressively higher magnifications. The fluorescence
micrographs confirm the accumulation of recombinant proteins in wheat embryo cells. Higher expressions of
recombinant proteins were observed in wheat embryo than endosperm cells. Bars 200 µm (10X), 100 µm
(20X), 50µm (40X), 50µm (63X).
61
III.6. Model fusion protein in wheat tissues
After successful transgenic plant generation, the expression and subcellular localization of
the recombinant fusion protein in wheat seed and vegetative tissues was investigated by
immunoblot (II.5.4), fluorescence (II.6.6) and confocal microscopy (II.6.7) and
immunolocalization experiments (II.6).
III.6.1. Presence and stability of the recombinant protein
The heterologous expression of repeated and Pro-X portion from γ-zein resulted in
ubiquitous expression the partner protein (DsReD) in a tissue independent manner in
wheat plant. Western blot analysis of wheat leaf, endosperm and embryo tissues extracts
(II.5.1) confirmed the integrity of the fused proteins, with an apparent molecular mass of
approximately 37 kDa (Fig.III.14 A). Total soluble proteins were extracted from wheat
tissues (II.5.1) separated by SDS-PAGE (II.5.2), transferred to a membrane (II.5.4) and
probed with a primary (rabbit anti-27kD γ-zein) and secondary antibody (goat anti-rabbit
IgG) and visualized using NBT/BCIP substrate. The presence of anticipated 37-kDa band
in all three tissues revealed that the recombinant fusion protein has been successfully
accumulated in wheat seed and vegetative tissues (Fig. III.14A). There was no equivalent
band in total soluble protein extracted (II. 5.1) from corresponding tissues in wild-type
wheat cv. Bobwhite. Total protein extracted (II.5.1) from transgenic tobacco leaves
expressing DsRed-zenH was used as a positive control.
To find out the possible degradation of the recombinant fusion protein in seed tissues
(endosperm, embryo), transgenic wheat seeds were stored for five months at normal room
conditions. The total proteins extracted (II.5.1) from wheat endosperm and embryo tissues
were subjected to SDS-PAGE (II.5.2) and western blot analysis (II.5.4). The protein
extracts were probed with the primary (rabbit anti-27kD γ-zein) and secondary (goat anti-
rabbit IgG) antibodies. The presence of 37-kDa bands presented the evidence that no
significant degradation in the endosperm and embryo tissues were detected (Fig.III.14B).
62
Figure III. 14. Immunoblot confirming the presence and stability of the recombinant γ-zein-DsRed
fusion protein in wheat tissues. Immunoblot confirms the accumulation of the recombinant γ-zein-DsRed
fusion proteins in different wheat tissues. Total soluble protein extracted from three wheat tissues (II, 5.1)
was separated by 12% (w/v) SDS-PAGE (II.5.2) and blotted onto a nitrocellulose membrane (II.5.4) for
immunodetection with antibodies against γ-zein. Visualization was performed using NBT/BCIP substrate.
Transgenic wheat seeds were stored for 5 months at room temperature. To find out possible degradation of
recombinant proteins, total protein extract from leaf, endosperm and embryo (II. 5.1) were separated by 12%
(w/v) SDS-PAGE (II.5.2) and blotted onto a nitrocellulose membrane (II.5.4) for immunodetection with
27kDa- γ-zein antibody and visualized using NBT/BCIP substrate.
A. Immunoblot micrograph for fresh wheat tissues extracts (II, 5.1).1. Pre-stained plus protein ladder. 2.
Positive control extract from transgenic tobacco expressing the DsRedzenH construct (II,5.1). 3. Negative
control extract from wild type wheat seed extract (II, 5.1) shows no band. 4. Young transgenic wheat leaf
extract (II, 5.1), containing the 37-kDa fusion protein. 5. Transgenic wheat endosperm extract (II, 5.1),
containing the recombinant fusion protein. 6. Transgenic wheat embryo extract (II, 5.1), containing the
recombinant fusion protein. B. The prescence of the recombinant fusion proteins (DsRed-zeinH) in wheat
seed tissues after five months storage. The existence of 37-kDa bands demonstrates that no degradation
products were detected in wheat tissues including 1. whole seed 2. embryo 3. endosperm.
63
III.6.2. Model fusion protein expression and accumulation in
transgenic wheat leaves
The detailed expression profile of the γ-zein-DsRed fusion protein in leaves was analyzed
by fluorescence (II.6.6) and confocal microscopy (II.6.7) revealing strong and constitutive
expression (Figures III.15A and III.11). The signal is clearly concentrated within small
protein aggregates approximately 2 µm in diameter, which is similar to the native zein
bodies in maize endosperm. The induced protein bodies in leaves appeared to be larger
than those induced in embryo cells. Figure III.15C shows leaf cross-sections stained with
anti-DsRed antibodies, revealing the anticipated formation of abundant and uniformly-
distributed protein bodies in the cytoplasm (Fig. III.15A, B). Immunolocalization
experiments showed that these recombinant DsRed bodies were always intracellular
(Fig.III.15C) and no signal was detected outside the cell wall confirmed that recombinant
fusion proteins were not secreted (Fig. III. 15C). Furthermore, no fluorescence was in the
negative control samples (wild type Bobwhite cv).
64
Figure III.15 DsRed protein bodies in leaf cells. Small squares of transgenic wheat leaf were fixed in 4%
(w/v) formaldehyde, 0.2% (w/v) glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (II.1.2). Microtome
sections were infiltrated and polymerised at 60˚C for 48 hours in LR White Resin. For immunolabeling,
ultrathin sections of 1-µm were obtained and incubated with primary antibody (DsRed polyclonal antibody)
at concentration of 1/500 at 4˚C overnight and secondary antibody (goat anti Rabbit, Alexa 546) at dilution
of 1/30 in 01 M phosphate buffer pH 7.4 (II.1.2) under dark conditions. For fluorescent (II.6.6) and confocal
microscopy analysis (II.6.7), the samples were cut in 0.1 M phosphate buffer pH 7.4 (II.1.2) and examined
under microscope.
A. Fluorescence micrograph (II, 6.6) of transgenic wheat leaf expressing recombinant fusion proteins which
are sequestered in cytosol. B. Confocal micrograph (II.6.7) of transgenic leaf expression numerous induced
protein bodies dispersed all over leaf cells, C. Immunofluorescence labeling (II, 6.5) of leaf ultra thin
sections showing the localization of recombinant protein bodies in leaf cells D. Fluorescent micrograph of
wild type wheat (Bobwhite) leaf where no red signals were observed. Bars 500 µm (A,B, D), 25 µm (C).
65
III.6.3. DsRed mobility in wheat leaf tissues
Time-lapse confocal imaging revealed that the fluorescent protein bodies were highly
dynamic, exhibiting unpredictable although unidirectional and continuous movement
within the leaf cells. This is the first time that the mobility of a recombinant protein has
been documented in wheat cells, although a similar phenomenon has been observed in
Nicotiana benthamiana leaves with a green fluorescent protein (GFP) elastin like peptide
(ELP) fusion (Conley et al., 2009). The significance of ceaseless moment of red bodies for
protein trafficking in wheat cells is unclear as they were moving within the cell without a
final destination. The precise intracellular pathway of the recombinant fusion protein
could not be predicted due to their unpredictable and unidirectional movement in each
cell.
III.6.4. Model fusion protein expression and accumulation in
transgenic wheat endosperm
The expression of γ-zein-DsRed fusion proteins in the endosperm cells was next
investigated. The recombinant fusion protein also induced protein bodies in the wheat
endosperm tissue. Fluorescence (II.6.6) and confocal microscopy (II.6.7) analyses
revealed the presence of numerous, evenly-distributed protein bodies containing DsRed,
but we observed a size gradient from the young endosperm cells below the aleurone layer
to the older cells in the deeper starchy endosperm 10 days post anthesis (Fig. III.16 A,C
and III.12). Figure III.17A and B show the presence of DsRed protein bodies in the
endosperm particularly in large vacuolar compartments containing aggregates of
endogenous storage protein bodies. The DsRed protein bodies can be seen as separate
peripheral entities in the prolamin bodies (Fig. III.16 C, B and III.17A,B). In addition, the
fluorescence signals of γ-zein-DsRed in endosperm cells appeared to be slightly weaker than
those observed in leaf and embryo cells. No labeling was detected in the apoplast or in any
other cell compartment such as starch granules (Fig. III.16B) and in the wild type wheat
endosperm. In the starchy endosperm cells, the artificial DsRed bodies were found spherical
structures of approximately 2-µm in size (Fig. III.16A), which strongly resemble in size and
appearance to the native zein bodies found in maize endosperm.
66
Immunolocalization experiments (II.6) of endosperm thin section tagged with polyclonal
DsRed antibody revealed the trafficking of recombinant proteins in the ectopic tissues. We
report that the recombinant fusion proteins in endosperm tissues were found localized in
artificial protein bodies. The recombinant DsRed bodies were either found separate
entities or sequestered into central PSVs (Fig. III.17A). In ectopic wheat endosperm cells,
the recombinant proteins most probably followed the bulk of endogenous glutenins. The
artificial protein bodies containing recombinant proteins could be seen in the cytoplasm
were apparently in the process of entering the central vacuole (arrow; Fig. III.17A). Figure
III. 16B demonstrate that the recombinant fusion protein has been localized in the central
PSV where recombinant γ-zein proteins could be found at the exterior of the central
storage vacuole (arrow). In transgenic wheat endosperm, the PSV contained two types of
prolamins, the native and recombinant fusion protein (maize prolamin). However, the
recombinant prolamins (γ-zein-DsRed) were distinguished by their fluorescence (Fig.
III.18A, B). Unlike leaf cells, the movement of the recombinant bodies could not be seen
in endosperm cell.
67
Figure III.16 Recombinant DsRed protein bodies in wheat endosperm. Small squares of transgenic
wheat endosperm were fixed in 4% (w/v) formaldehyde, 0.2% (w/v) glutaraldehyde in 0.1 M phosphate
buffer, pH 7.4 (II.1.2) as described (II.6.5). Specimen were infiltrated and polymerised at 60 ˚C for 48 hours
in LR White Resin. For immunolabeling, ultrathin microtome sections of 1-µm were obtained and incubated
with primary antibody (DsRed polyclonal antibody) at dilution of 1/500 and secondary antibody (goat anti
Rabbit, Alexa 546) at dilution of 1/30 in 01 M phosphate buffer (II.1.2) under dark conditions. For
fluorescent (II.6.6) and confocal microscopy (II.6.7), samples were cut in 0.1 M phosphate buffer pH 7.4
(II.1.2) and examined under microscope.
A. Fluorescence micrograph (II.6.6) of transgenic wheat endosperm expressing γ-zein-DsRed bodies
(arrows) are abundant in storage tissues. B. Immunofluorescence labeling (II.6.5) of transgenic wheat show
the locations of the tagged recombinant proteins in endosperm cells. DsRed bodies (arrows) are found
embedded in the protein bodies of wheat endosperm cells and at the periphery of storage vacuole (PSV). The
arrows indicate the recombinant protein accumulated in protein bodies within the PSV of wheat endosperm
cells C. Confocal microscopy (II.6.7) of transgenic wheat endosperm where DsRed bodies appear evenly
distributed within the endosperm. A closer view reveals that DsRed bodies are highly abundant in the seed
coat (sc) and also in the endosperm cells. Bars, 100 µm (A); 25 µm (B); 500 µm (C). Starch (s).
68
Figure III.17 Accumulation of recombinant DsRed protein bodies in transgenic wheat endosperm
cells. Small pieces of transgenic wheat endosperm were sliced in 0.1 M phosphate buffer, pH 7.4 (II.1.2)
and examined directly under a confocal microscope.
A Confocal images (II.6.7) show the accumulation of DsRed in the protein bodies in a large central protein
storage vacuole (PSV) of transgenic wheat endosperm cells (arrow). The induced protein bodies containing
recombinant proteins can also be seen in the cytoplasm (arrow). B. Corresponding transmission light image.
Bars, 50 µm (A and B)
III.6.5. Model fusion protein expression and accumulation in
transgenic wheat embryo
We report that engineered γ-zein portion also triggered the formation of artificial protein
bodies in wheat embryos cells. Fluorescence (II.6.6) and confocal images (II.6.7; Fig.
III.13; III.18 A, B) confirmed the well-define and highly fluorescent bodies distribution in
the embryo cells. Interestingly, the induced protein bodies were found to be smaller (~0.6
µm) than those found in endosperm and leaf tissues (Fig. III. 18A, B). Strong fluorescence
signals were observed in the tissues below the scutellum. However, the expression was not
equally distributed indicating that the ubiquitin promoter was not equally active in all
embryo tissues (Fig. III.18A). In comparison with endosperm cells, accumulation of
recombinant fusion proteins was recorded at earlier stages of seed development, eight days
post anthesis. The intensity of DsRed fluorescence also increased as the embryonic cells
matured.
69
The subcellular localization of recombinant fusion proteins in embryonic cells was also
identified. Immunofluorescence labeling (II.6.5) with primary antibody (anti-DsRed
polyclonal) followed by incubation with secondary antibody (goat anti rabbit, Alexa 546)
revealed the final destination of recombinant proteins in embryo cells. The ultra-thin
sections of the embryo showed the accumulation of recombinant proteins in the protein
bodies, those were found sequestered in the central storage vacuole (arrow; Fig. III.18 C).
The trafficking of recombinant proteins in embryonic tissues was consistent to those
observed in endosperm tissues.
70
Figure III.18 Accumulation of DsRed protein bodies in transgenic wheat embryos. Small squares of
transgenic wheat embryo were fixed in 4% (w/v) formaldehyde, 0.2% (w/v) glutaraldehyde in 0.1 M
phosphate buffer, pH 7.4 (II.1.2) as described (II.6.5). Specimen were infiltrated and polymerised at 60 ˚C
for 48 hours in LR White Resin. For immunolabeling (II.6.5), ultrathin microtome sections of 1-µm were
obtained and incubated with primary antibody (DsRed Polyclonal antibody) at dilution of 1/500 and
secondary antibody (goat anti Rabbit, Alexa 546) at dilution of 1/30 in 01 M phosphate buffer pH 7.4
(II.1.2) under dark conditions. For fluorescent (II.6.6) and confocal microscopy (II.6.7), samples were cut in
0.1 M phosphate buffer pH 7.4 and examined under microscope.
A. Fluorescence micrograph (II.6.6) Presence of numerous DsRed bodies (arrow) confirms the expression of
recombinant proteins in wheat embryo. However, in some tissues the recombinant expression is absent
(arrow). B. Confocal micrograph (II.6.7) shows the presence of recombinant DsRed bodies in wheat embryo
tissues. C. Immunofluorescence labeling (II.6.5) reveals the presence of abundant artificial protein
bodies in the embryo tissues aggregated at the periphery of the central PSV (arrow).
71
IV. DISCUSSION
Plants have been used for the experimental production of many different pharmaceutical
proteins, including vaccines (Streatfield, 2007), antibodies (Twyman et al. 2007),
hormones, signaling proteins, blood products and replacement enzymes (Stoger et al.
2002, 2005), and industrial enzymes (Fischer et al. 2003). As more recombinant proteins
are developed as industrial products, the demand increases for simple and economical
production platforms that allow the large-scale production of inexpensive heterologous
proteins. The advantages of plant-based production platforms have been widely
acknowledged, and although no single platform has emerged as a primary candidate for
process-scale manufacturing. Plant cell cultures offer the advantages of containment,
simplicity, consistency and simpler downstream processing, making them the most likely
platform in the short term to yield pharmaceutical products that comply with good
manufacturing practice (GMP). However, they suffer the same limitations in terms of
scalability and reliance on bioreactor-based infrastructure as other fermentation platforms.
Alternatively, transient expression systems offer the prospect of rapid and highly-scalable
production, but incapable of co-expressing two or more polypeptides, which are essential
for the assembly of heterooligomeric proteins. None of the plant-based production
platforms can yet challenge the yields possible using more established platforms such as
E. coli and mammalian cells (Ramessar et al., 2008) but their advantages in terms of
safety, scalability, economy and unique delivery mechanisms make it imperative to carry
out research for the improvement of productivity of plants for molecular farming. Among
plant expression systems, cereal crops are most attractive because seeds are potentially
one of the most economical systems for heterologous proteins (Kermode et al., 2012). The
recombinant proteins can be targeted to the naturally evolved protein storage organelles
such as ER derived protein bodies for protein function; prevent degradation and stable
accumulation for years (Ramessar et al., 2008). However, enhancing yield and efficient
methods for downstream processing of recombinant proteins are the greatest challenges
that must be overcome before seed based system can become as alternative for large-scale
production of heterologous proteins.
72
There are several reasons to select wheat plants as host for this study. Wheat has a well-
established global infrastructure for cultivation, harvest, storage and distribution,
providing the foundation for recombinant protein production in industrialized and
developing countries. Like other cereals, wheat seeds maintain recombinant proteins in a
stable form for years that makes it an attractive bioreactor for recombinant protein
production. Wheat has two major advantages over other crops such as maize in that it is
self-pollinating (thus reducing the likelihood of out-crossing from transgenic crops) and
has a low producer price compared to other cereals. As a cereal crop, it is generally
recognized as safe (GRAS) by the European Food Safety Authority (EFSA) and, therefore,
is an ideal production crop for oral vaccines and topical formulations. Potential
disadvantages of wheat include the challenging transformation procedure and the
generally low expression levels that have been achieved with other recombinant proteins,
hence the development of novel strategies to improve protein yields in this thesis. In
addition, the trafficking of recombinant proteins has been rarely studied in wheat seed
tissues. The recombinant protein production via γ-zein sequences and the insight of
heterologous protein trafficking in seed tissues would make it an attractive host for
molecular farming.
In this thesis, wheat seed tissues were chosen as preferred target for recombinant protein
production. This is because dry seeds provide an optimal environment for the stable
accumulation of recombinant proteins and are considered suitable as vehicles for
molecular farming (Stoger et al., 2002a). Seeds have been used for the production of many
recombinant proteins, including vaccine antigens, antibodies, hormones, proteases and
their inhibitors, growth factors and enzymes for industrial or medical applications (Boothe
et al., 2010). Targeting of recombinant proteins to naturally available storage organelles in
seed tissues assist higher accumulation and purification means for efficient production
system. Several publications have demonstrated convincingly that recombinant proteins in
mature seeds show no detectable loss of stability or activity when stored for several years
at ambient temperatures (Stoger et al. 2000, Nochi et al., 2007). In addition to stability,
high-yielding field-grown cereal crops also provide the benefit of scalability, simply by
planting more crops over a larger area.
73
In this study, wheat transformation was achieved by the particle bombardment using
immature embryos (III.2). This type of ex-plant was favored in early transformation
protocols (Becker et al., 1994; Rasco-Gaunt et al., 2001) although other targets have been
used for both particle bombardment and Agrobacterium-mediated transformation. One of
the key advantages of particle bombardment is that it is a genotype-independent method,
whereas Agrobacterium-mediated transformation must be optimized for individual
genotypes because of its high dependency on the biological properties of the target.
Particle bombardment is also suitable for the transient expression of endosperm (Knudsen
and Müller, 1991) or epidermal cells (Douchkov et al., 2005) to test the efficacy of
expression constructs. We achieved a low transformation frequency of up to < 1%, which
is comparable to early results reported by Altpeter et al. (1996) and is regarded as an
acceptable efficiency of gene transfer given the recalcitrance of wheat explants, the need
to culture explants on induction medium for several months and the challenge of
regenerating explants into transgenic plantlets (Stöger et al., 2000). The transformation
frequency is also dependent on the genotype, explant type and transformation
methodology, therefore, careful optimization was required to achieve this transformation
frequency in the Bobwhite cultivar.
In this thesis, we demonstrate the expression and sub-celullar accumulation of
recombinant proteins in three different wheat tissues by exploiting the natural ability of γ-
zein to induce the formation of artificial storage organelles in ectopic tissues. Our results
demonstrate that proline-rich repetitive (VHLPPP)8 and PX domains of maize prolamin
(γ-zein) are capable to trigger formation of artificial protein bodies in wheat seed and
vegetative tissues (III.6). In all tissues tested, the artificial protein bodies facilitated the
accumulation of recombinant proteins in dense protein bodies preventing them from
adverse enzymatic cell environment. The induced protein bodies in wheat tissues were
found as insoluble discrete spherical bodies that would allow their efficient and simple
isolation by low speed centrifugation. Our results illustrate that the γ-zein has maintained
the wild type characteristics of the native PBs in wheat tissues. Our findings suggest that
protein body formation and accumulation of recombinant protein in artificial 2-µm
spherical bodies is conserved in wheat leaf, endosperm and embryo tissues.
74
In maize (Zea mays), zeins are the major storage prolamins in the endosperm, and their
accumulation in zein bodies, derived from the endoplasmic reticulum, is well
characterized (Arcalis et al., 2010). Several zein classes (α, β, γ, δ), form large complexes
known as protein bodies within the ER-lumen. Interestingly, the 27 kDa γ-zein protein in
maize endosperm contains N-terminal signal peptide but retain intra-cellular in the ER
rather than being secreted. Moreover, γ-zein lacks the (H/K)DEL motif for ER retention
but localizes to the periphery of the protein bodies surrounding aggregates of other zein
proteins (Geli et al., 1994; Ludevid et al., 1994; α, β and δ-zein). When expressed
hetrologously, these gamma-zeins were able to trigger the formation of novel bodies both
in seed and vegetative tissues. However, the origin and mechanism by which γ-zein form
these novel cell organelles has not been elucidated. Nevertheless, experiments with the
mutated γ-zeins in Arabidopsis thaliana have provided evidence that both, the repeated
polypeptide and the C-terminal cystein rich domains, are responsible for protein bodies
induction, which may include protein-protein interactions or interactions with the ER
membranes (Geli et al., 1994). Various studies on zein accumulation in heterologous
expression systems suggest that the presence of γ-zein and β confers the stability of α and
δ zeins and facilitates their sequential assembly in the protein body in ER lumen. This
stabilizing effect of γ and β zein on the other zeins (α and δ) has been recognized in
transgenic tobacco and Arabidopsis plants (Bagga et al., 1997; Coleman et al 1996).
The heterologous expression of N-terminal proline rich domain of γ-zein result in the
ectopic formation of spherical structures suggests that some structural motifs are
responsible for de novo protein body formation (Saito et al., 2009). Two domains present
within γ-zein i.e. the repeat (PPPVHL)8 and Pro-X domains are involved in the ectopic
protein body formation. Whereas, repeated region of γ-zein is considered crucial for PB
biogenesis. The fusion protein lacking the repetitive portion (PPPVHL)8 to fluorescent
proteins resulted in the secretion of the fusion proteins but this repeat by itself did not
form PBs in tobacco vegetative tissues (Llop-Tous et al., 2010). More recently, the model
for Zera-induced PB biogenesis in tobacco leaves proposed by Llop-Tous and colleagues
(2010) highlighted the sequence elements responsible for protein body biogenesis.
According to their results, the eight repeat of (PPPVHL)8 is not only crucial for PB
formation but also important for the correct morphology of protein bodies. Shortening of
PPPVHL sequences (4-6) was still able to form PBs but comparatively smaller structures.
75
The presences of six Cystein residues present in repeated and PX domains are also vital
for protein biogenesis and may serve to stabilize the protein body assembly via
intermolecular disulfide bonds. Expression of zeolin in transgenic tobacco plant also
predicts the involvement of Cys residues for fusion protein polymerization and
accumulation in the ER (Pompa and Vitale 2006). Experiments with Cysteine residues in
Zera sequences explained that the two N-terminal Cys residues (Cys7 and Cys9) of Zera
are critical for oligomerization.
In this study, the expression of the recombinant fusion protein in wheat leaves was
confirmed with immunoblot analysis of young leaves extracts (III.14 A). The availability
of DsrRed bodies in these tissues was also validated with fluorescence and confocal
microscopy (III.15). In leaves, the fluorescent protein accumulated in ectopic protein
bodies 2 m in diameter which were dispersed throughout the cytoplasm (III.6.2). These
were similar in size and appearance to the native protein bodies found in maize
endosperm, and also to heterologous protein bodies induced in tobacco leaves using the N-
terminal γ-zein sequences (Torrent et al., 2009). In plants, the fusion of N-terminal
sequences of γ-zein (known as Zera®) with other proteins resulted in its accumulation in
novel protein bodies (Saito et al., 2009; Torrent et al., 2009a). Torrent and colleagues
(2009) fused the coding sequence for enhanced cyan fluorescent protein (ECFP) to either
the Zera domain or to an ER-retention signal (KDEL) and expressed the constructs in
tobacco epidermis. The Zera-ECFP protein accumulated in protein bodies within the
epidermal cells, the ECFP-KDEL protein accumulated in the ER lumen and a control
ECFP protein lacking Zera and KDEL was secreted. Similarly, the γ-zein N-terminal
sequences were also fused to the bean storage globulin phaseolin, and the recombinant
fusion protein (zeolin) was expressed in tobacco leaves where zeolin was accumulated in
the protein bodies (Mainieri et al. 2004). The same γ-zein N-terminal domain was unable
to increase yields of the recalcitrant HIV-1 Nef protein either as an N-terminal or C-
terminal fusion, but the entire zeolin protein as a fusion partner increased the yield of
zeolin-Nef to 1.5% total soluble protein and induced the formation of ectopic, ER-derived
protein bodies (de Virgilio et al., 2008). Llompart et al. (2010) produced human growth
hormone as a Zera fusion by transient expression in Nicotiana benthamiana leaves,
resulting in recombinant protein yields of up to 10% total soluble protein. Llop-Tous and
colleagues (2011) transiently produced an active insoluble aggregate of xylanase fused to
76
Zera in tobacco plant. Zera-xylanase fusion protein accumulated within ER-derived
protein bodies in leaves as active aggregates. That could later easily be extracted by a
simple density-based downstream process. Recently, Joseph and colleagues (2012)
utilized the zera sequences fused with DsRed fluorescent (Zera-DsRed) to target the fusion
partner into induced protein bodies and optimize the downstream purification of newly
formed PBs in Nicotiana benthamiana leaves. The fusion of zera sequences increased
accumulation of partner protein in induced protein bodies (up to 85% total TSP).
However, 195 additional proteins were also deposited in the Zera-DsRed polymer.
Interestingly, our results show that induced protein bodies resulted from model fusion
protein (γ-zein-DsRed) expression in leaf cells were observed as extremely mobile
organelles (III.6.3). To our knowledge, this study is the first to show recombinant protein
body mobility in wheat tissues. All the DsRed bodies were generally moving with the
same speed and transported in a unidirectional manner in each leaf cell. Experiments of
Conley and colleagues (2009) with ELP-GFP induced protein bodies are of great interest
to predict the factors affecting protein bodies mobility in Nicotiana benthamiana leaf
cells. The findings suggested that PB movement is dependent upon the integrity of actin
microfilaments and a functional actomyosin motility system (Conley et al., 2009). The
movement of our γ-zein-DsRed bodies in wheat cells are consistent with PBs movement
observed in tobacco epidermal cells where γ-gliadins-protein bodies like structure (PBLS)
were found highly mobile and were moving throughout the cells along with ER network
with no continuous direction. However, the movement observed under confocal
microscopy were concluded for possible correlation between PBLS size, movement, and
duration of expression. Furthermore, actin cytoskeleton dependent movement were also
verified in transgenic leaves (Francin-Allami et al., 2011). Recently, transient expression
of wheat γ-gliadin and low molecular weight (LMW) glutelin as fusion with green
fluorescent protein (GFP) in tobacco leaves formed protein body like structures (PBLS).
These spherical structures were also found highly mobile in tobacco leaves. The authors
explained the movement as actin-dependent (Francin-Allami et al., 2012). Above
mentioned experiments highlight that the mobility of ectopic protein bodies is not
restricted to certain plant species. Therefore, the involvement of cytockeleton in
trafficking of our recombinant γ-zein-DsRed bodies in transgenic wheat tissues cannot be
neglected and further investigations are highly recommended.
77
In addition to leaves, the recombinant fusion proteins were also expressed in wheat
endosperm and embryo cells to investigate the trafficking and accumulation of the protein
in seed tissues. This study is quite interesting to determine the impact of recombinant
protein expression in wheat endosperm and embryo, which forms its own endogenous
compartments as a major sink for storage proteins. We found that the recombinant fusion
protein (γ-zein-DsRed) was expressed in wheat seed tissues but that the expression profile
driven by the constitutive ubiquitin promoter was potentially constrained by the specific
cell type (III.6.4). In the endosperm, the highest expression was achieved in subaleurone
layer cells. This may reflect a genuine difference in the activity of the ubiquitin promoter
in different specialized cell types within the seed, but may also be explained by
differential activity in terms of protein synthesis and accumulation as the cells mature,
and/or the properties of the cells that affect the visibility of the fluorescence signal.
Subaleurone cells are younger than the central endosperm cells and generally contain more
protein bodies, whereas older cells contain more starch and the vacuole is reabsorbed.
Therefore, the visualization of protein bodies within the central vacuole is easier in the
subaleurone cells, where DsRed fluorescence could be detected from 10 to 25 days after
anthesis, peaking during days 14–18. The protein bodies appeared clustered or aggregated
in the central endosperm cells, perhaps reflecting a genuine difference in distribution
compared to leaves in combination with the clearer fluorescence signal in the larger
vacuoles of older cells. These results are in accordance with the behavior of endogenous
protein bodies, which also accumulate within large vacuoles. The γ-zein-DsRed protein
bodies in the inner endosperm cells were partially obscured by large starch granules.
Cell-specific differences in expression were also observed when we expressed the γ-zein-
DsRed fusion protein in wheat embryos (III.6.5). Strong fluorescence was observed in the
tissues below the scutellum, indicating that the ubiquitin promoter was not active in all of
the embryo tissues or that protein synthesis and accumulation were inefficient. At the level
of individual cells, the fusion protein appeared to be expressed more strongly in the
embryo than the endosperm. DsRed protein bodies were observed in clusters of cells
within the embryo from as early as 8 days after anthesis. However, the protein bodies in
embryo cells were less dynamic than those in leaf cells, as determined by real-time
confocal microscopy.
78
Degradation of recombinant proteins is critical because higher expression level does not
always insure the higher yield. Our recombinant fusion protein showed accumulation in all
three tissues tested. No degradation products were detected in wheat endosperm and
embryo tissues after five months (III.6.1). We suppose that the accumulation of
recombinant proteins in the novel protein bodies segregated the fusion protein away from
the secretory pathway (as no evidence of recombinant proteins in the apoplast) as well as
from ER associated degradation (ERAD) pathways (Benyair et al., 2011) and therefore
maintained the expression after five months. Similar to our results, Torrent and colleagues
(2009) also showed that the Zera sequences maintained the stability of the fusion partner,
allowing functional recombinant protein to be recovered after five months at room
temperature. The stability of proteins in protein bodies was also demonstrated by Nochi et
al. (2007), who expressed the cholera toxin B subunit in rice protein bodies and showed
they were highly resistant to proteolytic digestion in the gastrointestinal tract.
The expression and accumulation of the fusion protein in seed and vegetative tissues did
not appear to alter the growth or development of the transgenic wheat plants, neither in
terms of the general phenotype nor in terms of the impact at the cellular level. The
transgenic γ-zein-DsRed plants germinated were fertile and growth was identical to wild
type wheat plants. In another study (Torrent et al., 2009), there was also no evidence for
enhanced apoptosis in experiments using the Zera domain to express ECFP in tobacco
leaves. Also, targeting recombinant protein into induced protein bodies in cereal seed and
vegetative tissues with fluorescent protein GFP did not interfere with the plant growth and
development of tobacco plant (Saito et al., 2009). Other than plants, induced protein
bodies also did not alter growth when expressed in CHO and fungal cells (Torrent et al.,
2009). These results suggest that proline rich sequences of γ-zein when expressed as
fusion with other protein do not adversely affect plant growth.
In this thesis, we report the trafficking of recombinant fusion proteins in wheat seed and
vegetative tissues. In plant seed, enormous storage protein synthesis with diverse
subcellular destinations creates a logistic challenge for the endomembrane system. Protein
transport in cereal endosperm is complicated by the abundance of ER derived and vacuolar
protein bodies (Arcalis et al., 2004). In wheat seed tissues, prolamins are synthesized on
rER and then deposited to PSVs as their final destination. The movement from ER to PSV
79
occurs via two major transport routes, one bypassing and one passing through Golgi
complex. However, proteins travelling along each route merge at vacuole (Arcalis et al.,
2004). This final movement of storage proteins and their deposition into vacuole is still
controversial. Studies on protein trafficking in wheat demonstrate that prolamins assemble
in PBs within ER (Levanony et al, 1992; Rubin et al., 1992) and later bud off and
transported to vacuole by a process similar to autophagy (Galili et al., 1993). The role of
the Golgi apparatus has been controversial but generally, the Golgi independent pathway is
more widely accepted, although a Golgi-dependent pathway has also been proven for small
amounts of prolamins (Levanony et al., 1992). Golgi complex is the site for protein
glycosylation. In wheat seed tissues, prolamins are generally not glycosylated. However,
very low amount of glycosylation that may occur is very difficult to detect (Shewry, 1996).
In order to know how recombinant proteins respond to the wheat intracellular sorting
machinery, we investigated the trafficking of recombinant proteins in seed and leaf tissues.
For this reason, fluorescent reporter protein, DsRed was tracked for the final destination of
recombinant proteins. In vegetative tissues, the recombinant proteins were accumulated in
the novel protein bodies and were found dispersed in the cytoplasm (III.6.2).
Immunolocalization and fluorescent microscopic experiments confirmed that no ER or
apoplast accumulating signals were detected. These 2 µm red fluorescent bodies were
found highly mobile in leaf cells with no specific direction; however the movement were
recorded within the leave cells (III.6.3). Therefore, we conclude that the recombinant
proteins were targeted to novel protein bodies those were retained intracellularly in the
cytoplasm. In addition, the zigzag movement of recombinant bodies do not explain the
exact trafficking pattern in transgenic leaf cells.
The trafficking of the recombinant maize prolamins (γ-zeinDsRed) in the unique wheat
endosperm was of great interest in this thesis. Fluorescence, confocal and
immunofluorescent labelling experiments revealed that recombinant fusion proteins were
localized in artificial protein bodies and were found sequestered in the large protein
storage vacuoles which occupy the central part of both endosperm and embryo cells
(III.6.4; III.6.5). Furthermore, there was no trace of the molecule found in apoplast. In
wheat endosperm cells, the prolamins assemble in the protein bodies in the ER and
translocated to central PSVs in autophagy like process (Levanony et al., 1992). The
80
recombinant fusion protein bodies were found aggregated in the same compartment;
however they were distinguished with red fluorescence (III.6.4). In maize endosperm, the
vacuolar sequestration of prolamins has not been reported. We, therefore, conclude that
recombinant fusion protein followed the bulk of wheat endogenous glutenins in seed
tissues and deposited in the central PSV. Recombinant bodies were mostly found
aggregated at the periphery of the storage vacuole (PSV) in both endosperm and embryo
tissues (III.6.4 and III.6.5). In maize endosperm, four different types of zeins aggregate in
protein body where α-zein dominates the interior while γ-zeins are located at the periphery
(Coleman et al., 1999). The deposition of recombinant γ-zeinDsRed reflected the wild
type pattern but in different cell organelle and found at the exterior of central PSV in
wheat endosperm. The transgenic wheat embryo tissues also accumulated numerous novel
protein bodies. These artificial bodies were found aggregated in the central vacuole
(III.6.5). Different in expression of recombinant proteins were observed in transgenic
wheat endosperm and embryo tissues, however, the trafficking of recombinant proteins in
both tissues were found similar, accumulating in the central PSV as their final destination.
Our results are consistent with Coleman and his colleagues (2004), where heterologous
expression of zein in tobacco seed resulted in its sequestration into PSVs (Coleman et al.,
2004). Similarly, Bagga and colleagues (1995) observed the localization of 15kDa zein in
protein bodies sequestered into PSVs in tobacco seeds. These protein inclusions in the
seeds and leaves were also surrounded by membrane suggesting their derivation from the
ER. In wheat, recombinant proteins seem to favour their accumulation into PSVs in seeds
and follow the pathway exists for endogenous wheat prolamins in seed tissues. Also, the
recombinant protein (human serum albumin) containing a signal peptide was sequestered
into PBs and deposited into PSVs in wheat endosperm cells (Arcalis et al., 2004). Whereas
the same protein showed secretion when expressed in tobacco leaves (Verwoerd et al,
1995) and M. Truncatula seed cotyledons (Abranches et al., 2008). Similarly,
immunoglobulin compounds expressed for secretion in maize and rice endosperm was
accumulated in ER derived PBs and in PSVs (Nicholson et al, 2005).
We report that the combination of maize (γ-zein-DsRed) and native prolamins in seed
tissues did not alter cell functionality. Our results also report that fluorescent fusion
proteins are useful reporter molecules for confirmation of trafficking pathways. This is
81
because they could not only be tagged in immunolocalization experiments but also were
tracked with their fluorescence. These finding are of practical importance in molecular
farming applications, where the final destination of the heterologous proteins may
influence the activity, functionality and productivity.
Molecular farming initially focused on upstream productivity by improving gene
expression and protein stability, but as more products have moved towards commercial
development the focus has shifted to downstream processing for process-scale
manufacturing. The aggregation of the γ-zein-DsRed fusion protein into dense protein
bodies would simplify the purification process by allowing initial recovery by crude
fractionation, followed by the re-solubilization of relatively pure protein aggregates which
means that expensive chromatography-based separations are only needed for polishing to
remove trace impurities (Llop-Tous 2011). The accumulation of recombinant proteins in
novel protein bodies will therefore lower production costs by increasing upstream
productivity and by replacing expensive downstream processing steps (Joseph et al.,
2012).
This thesis also presents a protein fusion-based strategy to simplify the recombinant
protein purification via creation of artificial organelles. Although, the molecular farming
can be cheaper alternative to conventional platforms, however, downstream processing is
economically crucial because it represents 80 % of overall production cost (Fischer et al.,
2012). The expression of recalcitrant recombinant proteins as fusions with stability-
inducing partners is a generally useful strategy to improve product accumulation and to
assist in their subsequent purification (Conley et al., 2011; Llop-Tous et al., 2011).
However, the use of seed storage proteins as stabilizing partners provides additional
advantages including the induction of protein body-like aggregates in cells that normally
lack such organelles, offering further advantages such as protection from degradation,
alternative purification strategies and post-harvest encapsulation. The advantages of seed-
based systems can therefore be combined with the rapid biomass accumulation that is
possible in leafy plants. The protein-body inducing fusion partner that provide these
enhanced benefits include natural zeins, synthetic elastin-like peptides (ELPs) and fungal
hydrophobins (Table 2), all three of which can induce protein bodies and facilitate the
process-scale production and purification of recombinant proteins (Conley et al., 2011).
82
Both in plant and non-plant hosts, zein fusion strategy resulted in dense protein bodies and
favour the simple recovery by low speed centrifugation. In comparison, protein bodies
formed by the other two fusion partners were not dense and therefore the downstream
processing required transition cycling (ELP) and two phase procedure (Hydrophobin;
Llop-Tous et al., 2011)
ELPs are biochemically similar to γ-zein, and have been used to test the accumulation of
GFP targeted to the cytoplasm, plastids, apoplast and ER in several different N.
benthamiana tissues, with the ER variants showing the highest accumulation (Colney et
al., 2009). ELPs have also been used to increase the expression of pharmaceutical proteins
in transgenic tobacco leaves (Patel et al. 2007; Floss et al., 2008). The induction of protein
bodies using 100 ELP repeats fused to single-chain antibody resulted in yields of 40%
total soluble protein in tobacco seeds and 25% in leaves (Scheller et al., 2006).
Hydrophobins are small (ca. 10 kDa) and surface-active proteins derived from filamentous
fungi and can be either secreted or remain intracellular. When targeted to the ER,
recombinant hydrophobin fusion proteins also induce the formation of novel protein
bodies in fungi (Conley et al., 2011) and in tobacco leaves (Joensuu et al., 2010). In the
latter study, GFP was expressed using the Trichoderma reesei HFBI hydrophobin
sequence as a fusion partner, resulting in an unprecedented expression level equivalent to
51% total soluble protein in leaves, up to 91% of which could be recovered by aqueous
two-phase extraction (Joensuu et al., 2010).
The γ-zein fusion strategy was envisaged to produce stable vaccine antigens as prolamin
aggregates in wheat, but we found instead that separate fusion protein bodies were formed
and deposited in the PSV, which would also be useful for the development of seed-based
vaccines. Cold chains are usually required for vaccine distribution which is challenging in
developing countries, but seed-based oral vaccines provide a solution by allowing the
distribution of stable vaccine antigens at ambient temperatures using existing distribution
infrastructures for food (Ramessar et al., 2008). Edible seeds containing oral vaccines
would require minimal processing (enough to ensure dosing accuracy) and this could be
done by traditional techniques in developing country settings, such as the preparation of
flour paste (Lamphear et al., 2002; Takagi et al., 2005). Alvarez et al. (2010) expressed
Yersinia pestis F1-V antigen as a Zera fusion in Nicotiana benthamiana, Medicago sativa
83
(alfalfa) and Nicotiana tabacum (tobacco) NT1 cells. The yield was increased three-fold
compared to the native protein without affecting plant growth and development (Alvarez
et al., 2010).
Mucosal surface is the main entry source for many pathogens into the body; therefore,
both passive and active immunization approaches targeted to the mucosa have been
developed, with many researchers focusing on oral delivery (Hefferon et al., 2010). In
plants, the production of vaccine antigens have clear advantages because plant tissues
containing vaccine antigens can directly be consumed without processing avoiding costly
purification processes. However, the potential hurdle is the digestive system that may
cause destruction of the vaccine antigens before they can reach the immune cell in the
ileum, clustered in regions know as Peyer‟s patches. The production of vaccine antigens in
plants offers further advantage of extended partial protection from digestive enzymes and
permits more time for the antigen to interact with immune cells. This shielding effect is
improved through the targeting recombinant proteins into storage organelles such as
protein bodies (Takagi et al, 2010) and starch granules (reviewed in Khan et al., 2012;
Stephen et al, 2006).
Remarkably, Takagi and colleagues (2010) compared the survival of a known oral
tolerogen when administered either as a synthetic peptide, or as transgenic rice grain, in
which the antigen was targeted either to ER-derived protein bodies or PSVs. The rice
endosperm derived antigens showed more resistance to in vitro digestion with pepsin than
the soluble form. This is particularly noteworthy that antigens targeted to protein bodies
were more effective provided more resistance than PSVs. Therefore, the data presented the
evidence that bio-encapsulated delivery of tolerogen significantly enhanced the
immunological efficacy for the suppression of allergen-specific IgE responses. Ogawa and
colleagues (1987) also showed that rice PBs are partially digested and many protein
bodies were excreted. More recently, cholera toxin B subunit (Nochi et al., 2007) and
recombinant antibodies expressed in pea seeds (Zimmermann et al., 2009). In the latter
case, flour from the transgenic pea seeds was more potent than the purified antibody
fragments. Furthermore, the recombinant antibodies showed no detectable degradation
products in pea seed, possibly reflecting the existence of protease inhibitors (reviewed in
Khan et al., 2012; Zimmermann et al., 2009).
84
V. Conclusion
In this thesis, we show that a fusion strategy can be very efficient at producing
recombinant proteins in wheat plants. We report the introduction of exogenous organelles
in wheat seed and non-seed tissues using the maize prolamin γ-zein sequences as a fusion
for long-lasting production of recombinant proteins. Our results show that the addition of
proline rich polypeptide of maize prolamin to DsRed maintained the main characteristics
of wild-type γ-zein, resulted in the ubiquitous formation of PB-like structures and their
accumulation in PSVs in diverse wheat seed tissues. The results explain the encapsulation
of recombinant proteins into protein bodies generally enhances/maintain the stability both
in planta and post-harvest. This approach could therefore be implemented for the proteins
which are poorly secreted or degraded within the proteolytic cell conditions. Therefore,
the extension of our study to other cereals and proteins would be very interesting.
Knowledge of recombinant protein trafficking in wheat seed is more likely to be useful for
molecular farming applications.
85
VI. References
Abhary, M., Siritunga, D., Stevens, G., Taylor, N. J., and Fauquet, C. M. (2011).
Transgenic biofortification of the starchy staple cassava (Manihot esculenta) generates a
novel sink for protein. PLoS ONE 6, e16256.
Altpeter, F., Vasil, V., Srivastava, V., Stoger, E., Vasil, IK. (1996). Accelerated
production of transgenic wheat (Triticum aestiuum L.) plants. Plant Cell Rep 16, 12-17.
Alvarez, M., Topal, E., Martin, F., and Cardineau, G. (2010). Higher accumulation of F1-
V fusion recombinant protein in plants after induction of protein body formation. Plant
Mol Biol 72, 75-89.
Ao, Z., and Jane, J. l. (2007). Characterization and modeling of the A- and B-granule
starches of wheat, triticale, and barley. Carbo Poly 67, 46-55.
Arcalis, E., Marcel, S., Altmann, F., Kolarich, D., Drakakaki, G., Fischer, R., Christou, P.,
and Stoger, E. (2004). Unexpected deposition patterns of recombinant proteins in post-
endoplasmic reticulum compartments of wheat endosperm. Plant Physiol 136, 3457-3466.
Arcalis, E., Stadlmann, J., Marcel, S., Drakakaki, G., Winter, V., Rodriguez, J., Fischer,
R., Altmann, F., and Stoger, E. (2010). The changing fate of a secretory glycoprotein in
developing maize endosperm. Plant Physiol 153, 693-702.
Bagga, S., Adams, H., Kemp, J. D., and Sengupta-Gopalan, C. (1995). Accumulation of
15-kilodalton zein in novel protein bodies in transgenic tobacco. Plant Physiol 107, 13-23.
Bagga, S., Adams, H. P., Rodriguez, F. D., Kemp, J. D., and Sengupta-Gopalan, C.
(1997). Coexpression of the maize delta-zein and beta-zein genes results in stable
accumulation of delta-zein in endoplasmic reticulum-derived protein bodies formed by
beta-zein. Plant Cell 9, 1683-1696.
Becker, D., Brettschneider, R., and Lörz, H. (1994). Fertile transgenic wheat from
microprojectile bombardment of scutellar tissue. The Plant J 5, 299-307.
86
Benyair, R., Ron, E., Lederkremer, G. (2011). Protein quality control, retention, and
degradation at the endoplasmic reticulum. Int Rev Cell Mol Biol, 292:197-280.
Boothe, J., Nykiforuk, C., Shen, Y., Zaplachinski, S., Szarka, S., Kuhlman, P., Murray, E.,
Morck, D., and Moloney, M. M. (2010). Seed-based expression systems for plant
molecular farming. Plant Biotech J 8, 588-606.
Boothe, J. G., Saponja, J. A., and Parmenter, D. L. (1997). Molecular farming in plants:
Oilseeds as vehicles for the production of pharmaceutical proteins. Drug Devel Res 42,
172-181.
Breiteneder, H., and Mills, E. N. C. (2005). Plant food allergens structural and functional
aspects of allergenicity. Biotechnol Adv 23, 395-399.
Buléon, A., Colonna, P., Planchot, V., and Ball, S. (1998). Starch granules: structure and
biosynthesis. Inter J of Biol Macro 23, 85-112.
C. Neal, S., Jr. (2005). Monitoring the presence and expression of transgenes in living
plants. Trends in Plant Sci 10, 390-396.
C. Neal, S., Jr. (2006). Go with the glow: fluorescent proteins to light transgenic
organisms. Trends in Biotechnol 24, 155-162.
Canizares, M. C., Nicholson, L., and Lomonossoff, G. P. (2005). Use of viral vectors for
vaccine production in plants. Immunol Cell Biol 83, 263-270.
Chaudhary, S., Parmenter, D. L., and Moloney, M. M. (1998). Transgenic brassica
carinata as a vehicle for the production of recombinant proteins in seeds. Plant Cell Rep
17, 195-200.
Chikwamba, R. K., Scott, M. P., Mejía, L. B., Mason, H. S., and Wang, K. (2003).
Localization of a bacterial protein in starch granules of transgenic maize kernels. PNAS
100, 11127-11132.
Coleman, C. E., Herman, E. M., Takasaki, K., and Larkins, B. A. (1996). The maize
gamma-zein sequesters alpha zeins and stabilizes its accumulation in protein bodies of
transgenic tobacco endosperm. Plant Cell 8, 2335-2345.
87
Conley, A., Joensuu, J., Menassa, R., and Brandle, J. (2009a). Induction of protein body
formation in plant leaves by elastin-like polypeptide fusions. BMC Biol 7, 48.
Conley, A. J., Joensuu, J. J., Jevnikar, A. M., Menassa, R., and Brandle, J. E. (2009b).
Optimization of elastin-like polypeptide fusions for expression and purification of
recombinant proteins in plants. Biotechnol Bioeng 103, 562-573.
Conley, A. J., Joensuu, J. J., Richman, A., and Menassa, R. (2011). Protein body-inducing
fusions for high-level production and purification of recombinant proteins in plants. Plant
Biotechnol J 9, 419-433.
Conley, A. J., Mohib, K., Jevnikar, A. M., and Brandle, J. E. (2009c). Plant recombinant
erythropoietin attenuates inflammatory kidney cell injury. Plant Biotechnol J 7, 183-199.
Conrad, U., and Fiedler, U. (1998). Compartment-specific accumulation of recombinant
immunoglobulins in plant cells: an essential tool for antibody production and
immunomodulation of physiological functions and pathogen activity. Plant Mol Biol 38,
101-109.
Cunha, N., Murad, A., Ramos, G., Maranhão, A., Brígido, M., Araújo, A., Lacorte, C.,
Aragão, F., Covas, D., Fontes, A., Souza, G., Vianna, G., and Rech, E. (2011).
Accumulation of functional recombinant human coagulation factor IX in transgenic
soybean seeds. Trans Res 20, 841-855.
Dahleen, L., and Manoharan, M. (2007). Recent advances in barley transformation. In
Vitro Cel & Dev Biol Plant 43, 493-506.
de Virgilio, M., De Marchis, F., Bellucci, M., Mainieri, D., Rossi, M., Benvenuto, E.,
Arcioni, S., and Vitale, A. (2008). The human immunodeficiency virus antigen Nef forms
protein bodies in leaves of transgenic tobacco when fused to zeolin. J of Exper Bot 59,
2815-2829.
Ding, S.-H., Huang, L.-Y., Wang, Y.-D., Sun, H.-C., and Xiang, Z.-H. (2006). High-level
expression of basic fibroblast growth Factor in transgenic soybean seeds and
characterization of its biological activity. Biotechnol Lett 28, 869-875.
88
Douchkov, D., Nowara, D., Zierold, U., and Schweizer, P. (2005). A high-throughput
gene-silencing system for the functional assessment of defense-related Genes in barley
epidermal cells. Mol Plant-Micr Inter 18, 755-761.
Downing, W. L., Galpin, J. D., Clemens, S., Lauzon, S. M., Samuels, A. L., Pidkowich,
M. S., Clarke, L. A., and Kermode, A. R. (2006). Synthesis of enzymatically active human
α-l-iduronidase in Arabidopsis cgl (complex glycan-deficient) seeds. Plant Biotechnol J 4,
169-181.
Drakakaki, G., Marcel, S., Arcalis, E., Altmann, F., Gonzalez-Melendi, P., Fischer, R.,
Christou, P., and Stoger, E. (2006). The intracellular fate of a recombinant protein is tissue
dependent. Plant Physiol 141, 578-586.
Dus Santos, M. Ì. a. J., Wigdorovitz, A. s., Trono, K., Ros, R. l. D., Franzone, P. M., Gil,
F., Moreno, J., Carrillo, C., Escribano, J. M., and Borca, M. V. (2002). A novel
methodology to develop a foot and mouth disease virus (FMDV) peptide-based vaccine in
transgenic plants. Vaccine 20, 1141-1147.
Erlendsson, L. S., Muench, M. O., Hellman, U., Hrafnkelsdóttir, S. M., Jonsson, A.,
Balmer, Y., Mäntylä, E., and Örvar, B. L. (2010). Barley as a green factory for the
production of functional Flt3 ligand. Biotechnol J 5, 163-171.
Fiedler, U., and Conrad, U. (1995). High-level production and long-term storage of
engineered antibodies in transgenic tobacco seeds. Biotechnol (N Y) 13, 1090-1093.
Fischer, R., Liao, Y. C., Hoffmann, K., Schillberg, S., and Emans, N. (1999). Molecular
Farming of Recombinant Antibodies in Plants. Biol Chem 380, 825-839.
Fischer, R., Schillberg, S., Hellwig, S., Twyman, R. M., and Drossard, J. (2012). GMP
issues for recombinant plant-derived pharmaceutical proteins. Biotechnol Adv 2, 434-439.
Fischer, R., Stoger, E., Schillberg, S., Christou, P., and Twyman, R. M. (2004). Plant-
based production of biopharmaceuticals. Curr Opin Plant Biol 7, 152-158.
Fischer, R., Twyman, R. M., and Schillberg, S. (2003). Production of antibodies in plants
and their use for global health. Vaccine 21, 820-825.
89
Floss, D. M., Falkenburg, D., and Conrad, U. (2007). Production of vaccines and
therapeutic antibodies for veterinary applications in transgenic plants: an overview. Trans
Res 16, 315-332.
Floss, D. M., Sack, M., Stadlmann, J., Rademacher, T., Scheller, J., Stoger, E., Fischer, R.,
and Conrad, U. (2008). Biochemical and functional characterization of anti-HIV antibody-
ELP fusion proteins from transgenic plants. Plant Biotechnol J 6, 379-391.
Foresti, O., De Marchis, F., de Virgilio, M., Klein, E. M., Arcioni, S., Bellucci, M., and
Vitale, A. (2008). Protein domains involved in assembly in the endoplasmic reticulum
promote vacuolar delivery when fused to secretory GFP, indicating a protein quality
control pathway for degradation in the plant vacuole. Mol Plant 1, 1067-1076.
Foresti, O., Frigerio, L., Holkeri, H., de Virgilio, M., Vavassori, S., and Vitale, A. (2003).
A phaseolin domain involved directly in trimer assembly is a determinant for binding by
the chaperone BiP. Plant Cell 15, 2464-2475.
Francin-Allami, M., Axelle, Bouder., and Popineau, Y. (2012).Comparative study of
wheat low-molecular-weight glutenin and α-gliadin trafficking in tobacco cells. Plant Cell
Rep DOI: 10.1007/s00299-012-1343-8.
Francin-Allami, M., Saumonneau, A., Lavenant, L., Bouder, A., Sparkes, I., Hawes, C.,
Popineau, Y. (2011). Dynamic trafficking of wheat cgliadin and of its structural domains
in tobacco cells, studied with fluorescent protein fusions. J Exp Bot 62, 4507–4520.
Frigerio, L., Hinz, G., and Robinson, D. G. (2008). multiple vacuoles in plant cells: rule or
exception? Traffic 9, 1564-1570.
Frigerio, L., Pastres, A., Prada, A., and Vitale, A. (2001). Influence of KDEL on the fate
of trimeric or assembly-defective phaseolin: selective use of an alternative route to
vacuoles. Plant Cell 13, 1109-1126.
Fujiwara, Y., Aiki, Y., Yang, L., Takaiwa, F., Kosaka, A., Tsuji, N. M., Shiraki, K., and
Sekikawa, K. (2010). Extraction and purification of human interleukin-10 from transgenic
rice seeds. Prot Expr and Puri 72, 125-130.
90
Galili, G. (2004). ER-derived compartments are formed by highly regulated processes and
have special functions in plants. Plant Physiol 136, 3411-3413.
Galili, G., Altschuler, Y., and Levanony, H. (1993). Assembly and transport of seed
storage proteins. Trends in Cell Biol 3, 437-442.
Geli, M. I., Torrent, M., and Ludevid, D. (1994). Two structural domains mediate two
sequential events in gamma-zein targeting: Protein endoplasmic reticulum retention and
protein body formation. Plant Cell 6, 1911-1922.
Goff, S. A., Ricke, D., Lan, T.-H., Presting, G., Wang, R., Dunn, M., Glazebrook, J.,
Sessions, A., Oeller, P., Varma, H., Hadley, D., Hutchison, D., Martin, C., Katagiri, F.,
Lange, B. M., Moughamer, T., Xia, Y., Budworth, P., Zhong, J., Miguel, T., Paszkowski,
U., Zhang, S., Colbert, M., Sun, W.-l., Chen, L., Cooper, B., Park, S., Wood, T. C., Mao,
L., Quail, P., Wing, R., Dean, R., Yu, Y., Zharkikh, A., Shen, R., Sahasrabudhe, S.,
Thomas, A., Cannings, R., Gutin, A., Pruss, D., Reid, J., Tavtigian, S., Mitchell, J.,
Eldredge, G., Scholl, T., Miller, R. M., Bhatnagar, S., Adey, N., Rubano, T., Tusneem, N.,
Robinson, R., Feldhaus, J., Macalma, T., Oliphant, A., and Briggs, S. (2002). A draft
sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92-100.
Guerrero-Andrade, O., Loza-Rubio, E., Olivera-Flores, T., Fehervari-Bone, T. s., and
Gomez-Lim, M. A. (2006). Expression of the newcastle disease virus fusion protein in
transgenic maize and immunological studies. Trans Res 15, 455-463.
Hadlington, J. L., and Denecke, J. (2000). Sorting of soluble proteins in the secretory
pathway of plants. Curr Opi in Plant Biol 3, 461-468.
Hamada, S., Ishiyama, K., Choi, S.-B., Wang, C., Singh, S., Kawai, N., Franceschi, V. R.,
and Okita, T. W. (2003a). The transport of prolamine RNAs to prolamine protein bodies
in living rice endosperm cells. The Plant Cell 15, 2253-2264.
Hamada, S., Ishiyama, K., Sakulsingharoj, C., Choi, S.-B., Wu, Y., Wang, C., Singh, S.,
Kawai, N., Messing, J., and Okita, T. W. (2003b). Dual regulated RNA transport pathways
to the cortical region in developing rice endosperm. The Plant Cell 15, 2265-2272.
91
Hara-Nishimura, I., Shimada, T., Hatano, K., Takeuchi, Y., and Nishimura, M. (1998).
Transport of storage proteins to protein storage vacuoles is mediated by large precursor-
accumulating vesicles. The Plant Cell 10, 825-836.
He, Y., Ning, T., Xie, T., Qiu, Q., Zhang, L., Sun, Y., Jiang, D., Fu, K., Yin, F., Zhang,
W., Shen, L., Wang, H., Li, J., Lin, Q., Sun, Y., Li, H., Zhu, Y., and Yang, D. (2011).
Large-scale production of functional human serum albumin from transgenic rice seeds.
PNAS 10.1073/pnas.1109736108
Hefferon, K. (2010). The mucosal immune response to plant-derived vaccines. Phar Res
27, 2040-2042.
Herman, E., and Schmidt, M. (2004). Endoplasmic reticulum to vacuole trafficking of
endoplasmic reticulum bodies provides an alternate pathway for protein transfer to the
vacuole. Plant Physiol 136, 3440-3446.
Herman, E. M. (2008). Endoplasmic reticulum bodies: solving the insoluble. Curr Opin
Plant Biol 11, 672-679.
Herman, E. M., and Larkins, B. A. (1999). Protein storage bodies and vacuoles. The Plant
Cell 11, 601-614.
Hiatt, A., Cafferkey, R., and Bowdish, K. (1989). Production of antibodies in transgenic
plants. Nature 342, 76-78.
Hinz, G., Hillmer, S., Baumer, M., and Hohl, I. (1999). Vacuolar storage proteins and the
putative vacuolar sorting receptor BP-80 exit the golgi apparatus of developing pea
cotyledons in different transport Vesicles. The Plant Cell 11, 1509-1524.
Hohl, I., Robinson, D. G., Chrispeels, M. J., and Hinz, G. (1996). Transport of storage
proteins to the vacuole is mediated by vesicles without a clathrin coat. J. of Cell Sci 109,
2539-2550.
Holm, P. B., Olsen, O., Schnorf, M., Brinch-Pedersen, H., and Knudsen, S. (2000).
Transformation of barley by microinjection into isolated zygote protoplasts. Trans Res 9,
21-32.
92
Horvath, H., Huang, J., Wong, O., Kohl, E., Okita, T., Kannangara, C. G., and von
Wettstein, D. (2000). The production of recombinant proteins in transgenic barley grains.
PNAS 97, 1914-1919.
Hsieh, K., and Huang, A. H. C. (2004). Endoplasmic reticulum, oleosins, and oils in seeds
and tapetum cells. Plant Physiol 136, 3427-3434.
Huang, A. H. C. (1996). Oleosins and oil bodies in deeds and other organs. Plant Physiol
110, 1055-1061.
Ibl, V., and Stoger, E. The formation, function and fate of protein storage compartments in
seeds. (2011) Protop 249, 1-14.
Jach, G., Binot, E., Frings, S., Luxa, K., and Schell, J. (2001). Use of red fluorescent
protein from Discosoma sp. (dsRED) as a reporter for plant gene expression. The Plant J
28, 483-491.
James, M. G., Denyer, K., and Myers, A. M. (2003). Starch synthesis in the cereal
endosperm. Cur Opi in Plant Bio 6, 215-222.
Jane ek, ., and ev k, J. (1999). The evolution of starch-binding domain. FEBS Letters
456, 119-125.
Janecek, S., Svensson, B., and Henrissat, B. (1997). Domain evolution in the α-Amylase
family. J of Mol Evo 45, 322-331.
Jefferson, R. A., Kavanagh, T. A., and Bevan, M. W. (1987). GUS fusions: beta-
glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO
J 6, 3901-7.
Ji, Q., Vincken, J.-P., Suurs, L. C. J. M., and Visser, R. G. F. (2003). Microbial starch-
binding domains as a tool for targeting proteins to granules during starch biosynthesis.
Plant Mol Bio 51, 789-801.
Jiang, L., Phillips, T. E., Hamm, C. A., Drozdowicz, Y. M., Rea, P. A., Maeshima, M.,
Rogers, S. W., and Rogers, J. C. (2001). The protein storage vacuole. The J of Cell Bio
155, 991-1002.
93
Joensuu, J. J., Brown, K. D., Conley, A. J., Clavijo, A., Menassa, R., and Brandle, J. E.
(2009). Expression and purification of an anti-Foot-and-mouth disease virus single chain
variable antibody fragment in tobacco plants. Trans Res 5, 685-96
Joensuu, J. J., Conley, A. J., Lienemann, M., Brandle, J. E., Linder, M. B., and Menassa,
R. (2010). Hydrophobin fusions for high-level transient protein expression and
purification in nicotiana benthamiana. Plant Physiol 152, 622-633.
Joseph, M., Ludevid, D., Torrent, M., Rofidal, V., Tauzin. M., Rossignol. M., and Peltier
J. (2012). Proteomic characterisation of endoplasmic reticulum-derived protein bodies in
tobacco leaves. BMC Plant Biol.12, 1471-2229
Kawakatsu, T., Hirose, S., Yasuda, H., and Takaiwa, F. (2010). Reducing rice seed storage
protein accumulation leads to changes in nutrient quality and storage organelle formation.
Plant Physiol 154, 1842-1854.
Kay, R., Chan, A., Daly, M., and McPherson, J. (1987). Duplication of CaMV 35S
promoter sequences creates a strong enhancer for plant genes. Science 236, 1299-1302.
Kermode, AR. (2012). Seed expression systems for molecular farming. In: Wang A, Ma S
(eds) Molecular farming in plants: recent advances and future prospects. Springer Science
Business Media, New York, 89–123
Khan, I., Twyman, M., Arcalis, E., and Stoger, E. (2012). Using storage organelles for the
accumulation and encapsulation of recombinant proteins. Biotechnol J 7, 1099–1108.
Knudsen, S., and Müller, M. (1991). Transformation of the developing barley endosperm
by particle bombardment. Planta 185, 330-336.
Kogan, M. J., Dalcol, I., Gorostiza, P., Lopez-Iglesias, C., Pons, M., Sanz, F., Ludevid, D.,
and Giralt, E. (2001). Self-assembly of the amphipathic helix (VHLPPP)8. A mechanism
for zein protein body formation. J Mol Biol 312, 907-913.
Kogan, M. J., Dalcol, I., Gorostiza, P., Lopez-Iglesias, C., Pons, R., Pons, M., Sanz, F.,
and Giralt, E. (2002). Supramolecular properties of the proline-rich gamma-zein N-
terminal domain. Biophys J 83, 1194-1204.
94
Kreis, M., Forde, B. G., Rahman, S., Miflin, B. J., and Shewry, P. R. (1985). Molecular
evolution of the seed storage proteins of barley, rye and wheat. J of Mol Biol 183, 499-
502.
Krishnan, H. B., Franceschi, V. R., and Okita, T. W. (1986). Immunochemical studies on
the role of the Golgi complex in protein-body formation in rice seeds. Planta 169, 471-
480.
Kühnel, B., Alcantara, J., Boothe, J., van Rooijen, G., and Moloney, M. (2003). Precise
and efficient cleavage of recombinant fusion proteins using mammalian aspartic proteases.
Prot Eng 16, 777-783.
Kumamaru, T., Ogawa, M., Satoh, H., Okita, T., and Olsen, O.-A. (2007). Protein body
biogenesis in cereal endosperms. Endosperm 8, 141-158.
Kusnadi, A. R., Hood, E. E., Witcher, D. R., Howard, J. A., and Nikolov, Z. L. (1998).
Production and purification of two recombinant proteins from transgenic corn. Biotechnol
Progr 14, 149-155.
Lau, O. S., and Sun, S. S. M. (2009). Plant seeds as bioreactors for recombinant protein
production. Biotechnol Adv 27, 1015-1022.
Le Gall, F., Kipriyanov, S. M., Moldenhauer, G., and Little, M. (1999). Di-, tri- and
tetrameric single chain Fv antibody fragments against human CD19: effect of valency on
cell binding. FEBS Lett 453, 164-168.
Le Gall, F., Reusch, U., Moldenhauer, G., Little, M., and Kipriyanov, S. M. (2004).
Immunosuppressive properties of anti-CD3 single-chain Fv and diabody. J Immunol Meth
285, 111-127.
Lending, C. R., Kriz, A. L., Larkins, B. A., and Bracker, C. E. (1988). Structure of maize
protein bodies and immunocytochemical localization of zeins. Protopl 143, 51-62.
Levanony, H., Rubin, R., Altschuler, Y., and Galili, G. (1992). Evidence for a novel route
of wheat storage proteins to vacuoles. J Cell Biol 119, 1117-1128.
95
Li, X., Wu, Y., Zhang, D. Z., Gillikin, J. W., Boston, R. S., Franceschi, V. R., and Okita,
T. W. (1993). Rice prolamin protein body biogenesis: A BiP-mediated process. Science
262, 1054-1056.
Lichty, J. J., Malecki, J. L., Agnew, H. D., Michelson-Horowitz, D. J., and Tan, S. (2005).
Comparison of affinity tags for protein purification. Pro Expr and Purif 41, 98-105.
Lin, M., Rose-John, S., Grotzinger, J., Conrad, U., and Scheller, J. (2006). Functional
expression of a biologically active fragment of soluble gp130 as an ELP-fusion protein in
transgenic plants: purification via inverse transition cycling. Biochem J 398, 577-583.
Linder, M. B., Qiao, M., Laumen, F., Selber, K., Hyytia, T., Nakari-Setala, T., and
Penttila, M. E. (2004). Efficient purification of recombinant proteins using hydrophobins
as tags in surfactant-based two-phase systems. Biochem 43, 11873-11882.
Llompart, B., Llop-Tous, I., Marzabal, P., Torrent, M., Pallissé, R., Bastida, M., Ludevid,
M. D., and Walas, F. (2010). Protein production from recombinant protein bodies. Pro
Biochem 45, 1816-1820.
Llop-Tous I., Ortiz, M., Torrent, M., Ludevid, MD. (2011). The expression of a xylanase
targeted to ER-protein bodies provides a simple strategy to produce active insoluble
enzyme polymers in tobacco plants. PLoS One 2011, 6: e19474.
Ma, J. K., Barros, E., Bock, R., Christou, P., Dale, P. J., Dix, P. J., Fischer, R., Irwin, J.,
Mahoney, R., and Pezzotti, M. (2005). Molecular farming for new drugs and vaccines.
Current perspectives on the production of pharmaceuticals in transgenic plants. EMBO
Rep 6, 593-599.
Ma, J. K. C., Drake, P. M. W., and Christou, P. (2003). The production of recombinant
pharmaceutical proteins in plants. Nat Rev Genet 4, 794-805.
Mainieri, D., Rossi, M., Archinti, M., Bellucci, M., De Marchis, F., Vavassori, S., Pompa,
A., Arcioni, S., and Vitale, A. (2004). Zeolin. A new recombinant storage protein
constructed using maize gamma-zein and bean phaseolin. Plant Physiol 136, 3447-3456.
96
Mas, P., Devlin, P. F., Panda, S., and Kay, S. A. (2000). Functional interaction of
phytochrome B and cryptochrome 2. Nature 408, 207-211.
Matheson, L. A., Hanton, S. L., and Brandizzi, F. (2006). Traffic between the plant
endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond. Curr Opin Plant
Biol 9, 601-609.
Matthew J, H. (2004). Control of storage-product synthesis in seeds. Curr Opi in Plant
Biol 7, 302-308.
Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M.
L., and Lukyanov, S. A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa
species. Nat Biotechnol 17, 969-973.
Menassa, R., Nguyen, V., Jevnikar, A., and Brandle, J. (2001). A self-contained system
for the field production of plant recombinant interleukin-10. Mol Breed 8, 177-185.
Menassa, R., Zhu, H., Karatzas, C. N., Lazaris, A., Richman, A., and Brandle, J. (2004).
Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field
production. Plant Biotechnol J 2, 431-438.
Menkhaus, T. J., Bai, Y., Zhang, C., Nikolov, Z. L., and Glatz, C. E. (2004).
Considerations for the recovery of recombinant proteins from plants. Biotechnol Progr 20,
1001-1014.
Meyer, D. E., and Chilkoti, A. (1999). Purification of recombinant proteins by fusion with
thermally-responsive polypeptides. Nat Biotechnol 17, 1112-1115.
Meyer, D. E., and Chilkoti, A. (2004). Quantification of the effects of chain length and
concentration on the thermal behavior of elastin-like polypeptides. Biomacro 5, 846-851.
Miflin, B. J., Burgess, S. R., and Shewry, P. R. (1981). The development of protein bodies
in the storage tissues. J of Exper Bot 32, 199-219.
Miki, B., and McHugh, S. (2004). Selectable marker genes in transgenic plants:
applications, alternatives and biosafety. J of Biotechnol 107, 193-232.
97
Mishra, S., Yadav, D. K., and Tuli, R. (2006). Ubiquitin fusion enhances cholera toxin B
subunit expression in transgenic plants and the plant-expressed protein binds GM1
receptors more efficiently. J of Biotechnol 127, 95-108.
Mohamed-Yasseen, Y., Barringer, S., Splittstoesser, W., and Costanza, S. (1994). The role
of seed coats in seed viability. The Bot Rev 60, 426-439.
Molina, A., Hervás-Stubbs, S., Daniell, H., Mingo-Castel, A. M., and Veramendi, J.
(2004). High-yield expression of a viral peptide animal vaccine in transgenic tobacco
chloroplasts. Plant Biotechnol J 2, 141-153.
Moravec, T., Schmidt, M. A., Herman, E. M., and Woodford-Thomas, T. (2007).
Production of Escherichia coli heat labile toxin (LT) B subunit in soybean seed and
analysis of its immunogenicity as an oral vaccine. Vaccine 25, 1647-1657.
Muench, D. G., Chuong, S. D. X., Franceschi, V. R., and Okita, T. W. (2000). Developing
prolamine protein bodies are associated with the cortical cytoskeleton in rice endosperm
cells. Planta 211, 227-238.
Muench, D. G., Wu, Y., Zhang, Y., Li, X., Boston, R. S., and Okita, T. W. (1997).
Molecular cloning, expression and subcellular localization of a BiP homolog from rice
endosperm tissue. Plant Cell Physiol 38, 404-412.
Müntz, K., Belozersky, M. A., Dunaevsky, Y. E., Schlereth, A., and Tiedemann, J. (2001).
Stored proteinases and the initiation of storage protein mobilization in seeds during
germination and seedling growth. J of Exper Bot 52, 1741-1752.
Nicholson, L., Gonzalez-Melendi, P., van Dolleweerd, C., Tuck, H., Perrin, Y., Ma, J. K.,
Fischer, R., Christou, P., and Stoger, E. (2005). A recombinant multimeric
immunoglobulin expressed in rice shows assembly-dependent subcellular localization in
endosperm cells. Plant Biotechnol J 3, 115-127.
Nochi, T., Takagi, H., Yuki, Y., Yang, L., Masumura, T., Mejima, M., Nakanishi, U.,
Matsumura, A., Uozumi, A., Hiroi, T., Morita, S., Tanaka, K., Takaiwa, F., and Kiyono,
H. (2007). Rice-based mucosal vaccine as a global strategy for cold-chain- and needle-free
vaccination. PNAS USA 104, 10986-10991.
98
Nykiforuk, C., Shewmaker, C., Harry, I., Yurchenko, O., Zhang, M., Reed, C., Oinam, G.,
Zaplachinski, S., Fidantsef, A., Boothe, J., and Moloney, M. (2012). High level
accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower
(Carthamus tinctorius) seeds. Trans Res 9, 1-15.
Obregon, P., Chargelegue, D., Drake, P. M. W., Prada, A., Nuttall, J., Frigerio, L., and
Ma, J. K. C. (2006). HIV-1 p24–immunoglobulin fusion molecule: a new strategy for
plant-based protein production. Plant Biotechnol J 4, 195-207.
Ow, D. W., De Wet, J. R., Helinski, D. R., Howell, S. H., Wood, K. V., and Deluca, M.
(1986). Transient and stable expression of the firefly luciferase gene in plant cells and
transgenic plants. Science 234, 856-859.
Parmenter, D. L., Boothe, J. G., Rooijen, G. J. H., Yeung, E. C., and Moloney, M. M.
(1995). Production of biologically active hirudin in plant seeds using oleosin partitioning.
Plant Mol Bio 29, 1167-1180.
Patel, J., Zhu, H., Menassa, R., Gyenis, L., Richman, A., and Brandle, J. (2007). Elastin-
like polypeptide fusions enhance the accumulation of recombinant proteins in tobacco
leaves. Trans Res 16, 239-249.
Patel, M., Johnson, J. S., Brettell, R. I. S., Jacobsen, J., and Xue, G.-P. (2000). Transgenic
barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol
Breed 6, 113-124.
Perrin, Y., Vaquero, C., Gerrard, I., Sack, M., Drossard, J. r., Stoger, E., Christou, P., and
Fischer, R. (2000). Transgenic pea seeds as bioreactors for the production of a single-
chain Fv fragment (scFv) antibody used in cancer diagnosis and therapy. Mol Breed 6,
345-352.
Persson, J., Nyström, L., Ageland, H., and Tjerneld, F. (1999). Purification of recombinant
and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems:
Recycling of thermoseparating polymer and surfactant with temperature-induced phase
separation. Biotechnol and Bioeng 65, 371-381.
99
Petruccelli, S., Otegui, M. S., Lareu, F., Tran Dinh, O., Fitchette, A. C., Circosta, A.,
Rumbo, M., Bardor, M., Carcamo, R., and Gomord, V. (2006). A KDEL-tagged
monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is
both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol J
4, 511-527.
Pompa, A., and Vitale, A. (2006). Retention of a bean phaseolin/maize gamma zein fusion
in the endoplasmic reticulum depends on disulfide bond formation. Plant Cell 18, 2608-
2621.
Prat, S., Cortadas, J., Puigdomenech, P., and Palau, J. (1985). Nucleic acid (cDNA) and
amino acid sequences of the maize endosperm protein glutelin-2. Nucl Acids Res 13,
1493-1504.
Qian, B., Shen, H., Liang, W., Guo, X., Zhang, C., Wang, Y., Li, G., Wu, A., Cao, K., and
Zhang, D. (2008). Immunogenicity of recombinant hepatitis B virus surface antigen fused
with preS1 epitopes expressed in rice seeds. Trans Res 17, 621-631.
Rademacher, T., Sack, M., Arcalis, E., Stadlmann, J., Balzer, S., Altmann, F., Quendler,
H., Stiegler, G., Kunert, R., Fischer, R., and Stoger, E. (2008). Recombinant antibody
2G12 produced in maize endosperm efficiently neutralizes HIV-1 and contains
predominantly single-GlcNAc N-glycans. Plant Biotechnol J 6, 189-201.
Ramessar, K., Capell, T., and Christou, P. (2008). Molecular pharming in cereal crops.
Phytochem Rev 7, 579-592.
Ramirez, N., Ayala, M., Lorenzo, D., Palenzuela, D., Herrera, L., Doreste, V., Perez, M.,
Gavilondo, J. V., and Oramas, P. (2002). Expression of a single-chain Fv antibody
fragment specific for the hepatitis B surface antigen in transgenic tobacco plants. Trans
Res 11, 61-64.
Randall, J., Bagga, S., Adams, H., and Kemp, J. D. (2000). A modified 10 kD zein protein
produces two morphologically distinct protein bodies in transgenic tobacco. Plant Sci 150,
21-28.
100
Randall, J. J., Sutton, D. W., Hanson, S. F., and Kemp, J. D. (2005). BiP and zein binding
domains within the delta zein protein. Planta 221, 656-666.
Rasco‐Gaunt, S., Riley, A., Cannell, M., Barcelo, P., and Lazzeri, P. A. (2001).
Procedures allowing the transformation of a range of European elite wheat (Triticum
aestivum L.) varieties via particle bombardment. J Exp Bot 52, 865-874.
Rubin, R., Levanony, H., and Galili, G. (1992). Evidence for the Presence of two different
types of protein bodies in wheat endosperm. Plant Physiol 99, 718-724.
Rymerson, R. T., Menassa, R., and Brandle, J. E. (2002). Tobacco, a platform for the
production of recombinant proteins. Molecular Farming of Plants and Animals for Human
and Veterinary Medicine, 1-32.
Saalbach, I., Giersberg, M., and Conrad, U. (2001). High-level expression of a single-
chain Fv fragment (scFv) antibody in transgenic pea seeds. J Plant Physiol 158, 529-533.
Saint-Jore, C. M., Evins, J., Batoko, H., Brandizzi, F., Moore, I., and Hawes, C. (2002).
Redistribution of membrane proteins between the Golgi apparatus and endoplasmic
reticulum in plants is reversible and not dependent on cytoskeletal networks. Plant J 29,
661-678.
Saito, Y., Kishida, K., Takata, K., Takahashi, H., Shimada, T., Tanaka, K., Morita, S.,
Satoh, S., and Masumura, T. (2009). A green fluorescent protein fused to rice prolamin
forms protein body-like structures in transgenic rice. J Exp Bot 60, 615-627.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning: A laboratory
manual. 2.
Sanford, J.C., Smith, F. D., and Russell, J.A. (1993). Optimizing the biolistic process.
Meth in Enzy 217, 483-509.
Scheller, J., Henggeler, D., Viviani, A., and Conrad, U. (2004). Purification of spider silk-
elastin from transgenic plants and application for human chondrocyte proliferation. Trans
Res 13, 51-57.
101
Scheller, J., Leps, M., and Conrad, U. (2006). Forcing single-chain variable fragment
production in tobacco seeds by fusion to elastin-like polypeptides. Plant Biotechnol J 4,
243-249.
Schillberg, S., Emans, N., and Fischer, R. (2002). Antibody molecular farming in plants
and plant cells. Phytochem Rev 1, 45-54.
Schillberg, S., Fischer, R., and Emans, N. (2003). Molecular farming of recombinant
antibodies in plants. Cell and Mol Life Sci 60, 433-445.
Schmidt, M. A., and Herman, E. M. (2008). Proteome rebalancing in soybean seeds can be
exploited to enhance foreign protein accumulation. Plant Biotechnol J 6, 832-842.
Schünmann, P. H. D., Coia, G., and Waterhouse, P. M. (2002). Biopharming the
SimpliRED™ HIV diagnostic reagent in barley, potato and tobacco. Mol Breed 9, 113-
121.
Selber, K., Tjerneld, F., Collen, A., Hyytia, T., Nakari-Setala, T., Bailey, M., Fagerstra,
R., Kan, J., van der Laan, J., Penttila, M., and Kula, M.-R. (2004). Large-scale separation
and production of engineered proteins, designed for facilitated recovery in detergent-based
aqueous two-phase extraction systems. Proc Biochem 39, 889-896.
Shewry, P. R., and Halford, N. G. (2002). Cereal seed storage proteins: structures,
properties and role in grain utilization. J Exp Bot 53, 947-958.
Shewry, P. R., Napier, J. A., and Tatham, A. S. (1995). Seed storage proteins: Structures
and biosynthesis. The Plant Cell 7, 945-956.
Singh, N. K., Shepherd, K. W., Langridge, P., and Gruen, L. C. (1991). Purification and
biochemical characterization of triticin, a legumin-like protein in wheat endosperm. J Cer
Sci 13, 207-219.
Skerra, A., and Schmidt, T. G. M. (2000). Use of the strep- tag and streptavidin for
detection and purification of recombinant proteins. Meth in Enzy 326, 271-304.
Smith, A. M. (2001). The biosynthesis of starch granules. Biomacro 2, 335-341.
102
Smith, M. L., Richter, L., Arntzen, C. J., Shuler, M. L., and Mason, H. S. (2003).
Structural characterization of plant-derived hepatitis B surface antigen employed in oral
immunization studies. Vaccine 21, 4011-4021.
Stöger, E., Ma, J. K., Fischer, R., and Christou, P. (2005a). Sowing the seeds of success:
Pharmaceutical proteins from plants. Curr Opin Biotechnol 16, 167-173.
Stöger, E., Sack, M., Fischer, R., and Christou, P. (2002). Plantibodies: applications,
advantages and bottlenecks. Curr Opin Biotechnol 13, 161-166.
Stöger, E., Sack, M., Nicholson, L., Fischer, R., and Christou, P. (2005c). Recent progress
in plantibody technology. Curr Pharm Des 11, 2439-2457.
Stöger, E., Vaquero, C., Torres, E., Sack, M., Nicholson, L., Drossard, J., Williams, S.,
Keen, D., Perrin, Y., Christou, P., and Fischer, R. (2000). Cereal crops as viable
production and storage systems for pharmaceutical scFv antibodies. Plant Mol Biol 42,
583-590.
Streatfield, S. J. (2007). Approaches to achieve high-level heterologous protein production
in plants. Plant Biotechnol J 5, 2-15.
Takagi, H., Hiroi, T., Hirose, S., Yang, L., and Takaiwa, F. (2010). Rice seed ER-derived
protein body as an efficient delivery vehicle for oral tolerogenic peptides. Peptides 31,
1421-1425.
Takaiwa, F., Hirose, S., Takagi, H., Yang, L., and Wakasa, Y. (2009). Deposition of a
recombinant peptide in ER-derived protein bodies by retention with cysteine-rich
prolamins in transgenic rice seed. Planta 229, 1147-1158.
Takaiwa, F., Takagi, H., Hirose, S., and Wakasa, Y. (2007a). Endosperm tissue is good
production platform for artificial recombinant proteins in transgenic rice. Plant Biotechnol
J 5, 84-92.
Takaiwa, F., Takagi, H., Hirose, S., and Wakasa, Y. (2007b). Endosperm tissue is good
production platform for artificial recombinant proteins in transgenic rice. Plant Biotechnol
J 5, 84-92.
103
Tanaka, A., Ito, M., and Hiromi, K. (1986). Equilibrium and kinetic studies on the binding
of gluconolactone to almond beta-glucosidase in the absence and presence of glucose. J of
Biochem 100, 1379-1385.
Terpe, K. (2003). Overview of tag protein fusions: from molecular and biochemical
fundamentals to commercial systems. App Micro and Biotechnol 60, 523-533.
Thorpe, J. (1999). The application of LR Gold resin for immunogold labeling. Methods in
Molecular Biology: Elec Micros Meth and Prot 117, 99-110.
Torrent, M., Geli, M. I., Ruiz-Avila, L., Canals, J. M., Puigdomenech, P., and Ludevid, D.
(1994). Role of structural domains for gamma zein retention in xenopus oocytes. Planta
192, 512-518.
Torrent, M., Llompart, B., Lasserre-Ramassamy, S., Llop-Tous, I., Bastida, M., Marzabal,
P., Westerholm-Parvinen, A., Saloheimo, M., Heifetz, P., and Ludevid, M. D. (2009a).
Eukaryotic protein production in designed storage organelles. BMC Biology 7, 5.
Torrent, M., Llompart, B., Lasserre-Ramassamy, S., Llop-Tous, I., Bastida, M., Marzabal,
P., Westerholm-Parvinen, A., Saloheimo, M., Heifetz, P. B., and Ludevid, M. D. (2009b).
Eukaryotic protein production in designed storage organelles. BMC Biol 7, 5.
Torrent, M., Llop-Tous, I., and Ludevid, D. M. (2009c). Protein body induction: a new
tool to produce and recover recombinant proteins in plants. Rec Pro from Plants, Meth in
Mol Biol 483, 193-208.
Tosi, P., Parker, M., Gritsch, C. S., Carzaniga, R., Martin, B., and Shewry, P. R. (2009).
Trafficking of storage proteins in developing grain of wheat. J Exp Bot 60, 979-991.
Tsien, R. Y. (1998). The green fluorescent protein. Annu Rev Biochem 67, 509-544.
Tsuboi, T., Takeo, S., Iriko, H., Jin, L., Tsuchimochi, M., Matsuda, S., Han, E.-T., Otsuki,
H., Kaneko, O., Sattabongkot, J., Udomsangpetch, R., Sawasaki, T., Torii, M., and Endo,
Y. (2008). Wheat germ cell-free system-based production of malaria proteins for
discovery of novel vaccine candidates. Infec and Immunol 76, 1702-1708.
104
Twyman, R. M., Schillberg, S., and Fischer, R. (2005). Transgenic plants in the
biopharmaceutical market. Exp Opin on Emerg Drugs 10, 185-218.
Twyman, R. M., Schillberg, S., Fischer, R., and Ranalli, P. (2007). Molecular farming of
antibodies in plants. Improvement of crop plants for industrial end uses. 435-469.
Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., and Fischer, R. (2003b).
Molecular farming in plants: host systems and expression technology. Trends Biotechnol
21, 570-578.
Urry, D. W. (1988). Entropic elastic processes in protein mechanisms. I. Elastic structure
due to an inverse temperature transition and elasticity due to internal chain dynamics. J of
Prot Chem 7, 1-34.
Van Droogenbroeck, B., Cao, J., Stadlmann, J., Altmann, F., Colanesi, S., Hillmer, S.,
Robinson, D. G., Van Lerberge, E., Terryn, N., Van Montagu, M., Liang, M., Depicker,
A., and Jaeger, G. D. (2007). Aberrant localization and underglycosylation of highly
accumulating single-chain Fv-Fc antibodies in transgenic Arabidopsis seeds. PNAS 104,
1430-1435.
Vasil, V., Castillo, A. M., Fromm, M. E., and Vasil, I. K. (1992). Herbicide resistant
fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable
embryogenic callus. Nat Biotechnol 10, 667-674.
Verwoerd, T., Paridon, P., Ooyen, A., Lent, J., Hoekema, A., Pen, J. (1995). Stable
accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol
109,1199–1205.
Vitale, A., and Ceriotti, A. (2004). Protein quality control mechanisms and protein storage
in the endoplasmic reticulum. A conflict of interests? Plant Physiol 136, 3420-3426.
Vitale, A., and Hinz, G. (2005). Sorting of proteins to storage vacuoles: how many
mechanisms? Trends in Plant Sci 10, 316-323.
105
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). An enhanced transient
expression system in plants based on suppression of gene silencing by the p19 protein of
tomato bushy stunt virus. Plant J 33, 949-956.
Washida, H., Sugino, A., Messing, J., Esen, A., and Okita, T. W. (2004). Asymmetric
localization of seed storage protein RNAs to distinct subdomains of the endoplasmic
reticulum in developing maize endosperm cells. Plant Cell Physiol 45, 1830-1837.
Wilson, S., Burton, R., Doblin, M., Stone, B., Newbigin, E., Fincher, G., and Bacic, A.
(2006). Temporal and spatial appearance of wall polysaccharides during cellularization of
barley (Hordeum vulgare) endosperm. Planta 224, 655-667.
Witte, C.-P., Noël, L., Gielbert, J., Parker, J., and Romeis, T. (2004). Rapid one-step
protein purification from plant material using the eight-amino acid StrepII epitope. Plant
Mol Biol 55, 135-147.
Woo, Y.-M., Hu, D. W.-N., Larkins, B. A., and Jung, R. (2001). Genomics analysis of
genes expressed in maize endosperm identifies novel seed proteins and clarifies patterns of
zein gene expression. The Plant Cell 13, 2297-2317.
Wu, J., Yu, L., Li, L., Hu, J., Zhou, J., and Zhou, X. (2007). Oral immunization with
transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces
protective immune responses in chickens. Plant Biotechnol J 5, 570-578.
Xie, T., Qiu, Q., Zhang, W., Ning, T., Yang, W., Zheng, C., Wang, C., Zhu, Y., and Yang,
D. (2008). A biologically active rhIGF-1 fusion accumulated in transgenic rice seeds can
reduce blood glucose in diabetic mice via oral delivery. Peptides 29, 1862-1870.
Yamagata, H., and Tanaka, K. (1986). The site of synthesis and accumulation of rice
storage proteins. Plant and Cell Physiol 27, 135-145.
Yang, J., Barr, L. A., Fahnestock, S. R., and Liu, Z.-B. (2005). High yield recombinant
silk-like protein production in transgenic plants through protein targeting. Trans Res 14,
313-324.
106
Yang, Y., Li, R., and Qi, M. (2000). In vivo analysis of plant promoters and transcription
factors by agroinfiltration of tobacco leaves. Plant J 22, 543-551.
Zakharov, A., Giersberg, M., Hosein, F., Melzer, M., Muntz, K., and Saalbach, I. (2004).
Seed-specific promoters direct gene expression in non-seed tissue. J Exp Bot 55, 1463-
1471.
Zhang, F., and Boston, R. S. (1992). Increases in binding protein (BiP) accompany
changes in protein body morphology in three high-lysine mutants of maize. Protoplasma
171, 142-152.
Zhang, Y., Darlington, H., Jones, H. D., Halford, N. G., Napier, J. A., Davey, M. R.,
Lazzeri, P. A., and Shewry, P. R. (2003). Expression of the gamma-zein protein of maize
in seeds of transgenic barley: effects on grain composition and properties. Theor and App
Gen 106, 1139-1146.
Ziemienowicz, A. (2001). Plant selectable markers and reporter genes. Acta Physiol Plant
23, 363-374.
Zimmermann, J., Saalbach, I., Jahn, D., Giersberg, M., Haehnel, S., Wedel, J., Macek, J.,
Zoufal, K., Glunder, G., Falkenburg, D., and Kipriyanov, S. (2009). Antibody expressing
pea seeds as fodder for prevention of gastrointestinal parasitic infections in chickens. BMC
Biotechnol 9, 79.
107
Appendix
List of Abbreviations
°C degree Celsius
µ micro
2,4-D 2,4-Dichlorophenoxyessigsäure
A. tumefaciens Agro-bacterium tumefaciens
aa amino acids
Ab antibody
Amp ampicillin
AP alkaline phosphatase
APO apoplast
BCIP 5-Bromo-4-Chloro-3-Indolyl-
Phosphat
bp base pair
CaCl2 calcium chloride
CaMV 35S 35S promoter from the cauliflower
mosaic virus
CaMV cauliflower mosaic virus
Carb carbenicillin
cDNA complementary DNA
CI chloroform/isoamyl alcohol (24:1)
cv. cultivar
Cys cysteine
DNA deoxyribonucleic acid
DNTP deoxyribonucleoside triphosphate
DsRed discosoma Red Flourescent Protein
E. coli Escherichia coli
e.g. exempli gratia
ER endoplasmic reticulum
EtBr ethidium bromide
EtOH ethanol
108
g relative centrifugal force (RCF)
GFP green fluorescent protein
GSM Agrobacteria glycerol stock media
h hour(s)
H or His6 six histidine residues tag
HCl hydrochloric acid
He helium
i.e. id est
kb kilobase pair
kDa kilodalton
KDEL KDEL (Lys-Asp-Glu-Leu) ER
retention signal
Km Kanamycin
L liter
LB Luria-Bertani medium
LBamp LB media containing ampicillin
m mili
M molar
M molar
min Minute(s)
mM millimolar
Mr Molecular mass
mRNA messenger RNA
MS Murashige and Skoog medium
Mw molecular weight
MW molecular weight
NA not available
NaCl nodium chloride
nd. not detectable
nt nucleotide
O/N over night
OD optical density
ORF open reading frame
109
Ori origin of replication
PAA poly-acrylamide
PAGE poly-acrylamide gel electrophoresis
PAT phosphinothricin acetyl transferase
pat-gene phosphinotricin-acetyltransferase
gene
PB protein bodies
PBS phosphate buffered saline
PBST 0.1% (v/v) Tween-20 in PBS
PCR polymerase chain reaction
PEG polyethylene glycol
pH A logarithmic measure of hydrogen
ion concentration
poly A polyadenylation signal
PPT phosphinothricin
psi pound per square inch
PSV protein storage vacuole
rec. recombinant
rHSA recombinant human serum albumin
RNA Ribonucleic acid
rpm Rounds per minute
RT room temperature
SDS-PAGE Sodium dodecyl sulfate-
polyacrylamide gel electrophoresis
sec second(s)
Taq Thermus aquaticus
TEMED N, N, N, N-tetramethylene-
ethylenediamine
temp. temperature
Tm melting temperature
TSP total soluble protein.
Tris Tris (Hydroxymethyl) aminomethane
110
Tween 20 polyoxyethylene sorbitan
monolaureate
ubi1-Promotor ubiquitin1-Promotor
UTR untranslated region
UV ultraviolet
UV ultraviolet light
v/v volume per volume
Vol. volume
w/v weight per volume
w/w weight per weight
α Apha
β Beta
γ Gamma
μg microgram
μl microliter
μm micrometer
μM micromolar
111
Curriculum Vitae
Name: Imran Khan
Gender: Male
Date of Birth: 1st April, 1980
Place of Birth: Peshawar, Pakistan
Nationality: Pakistani
Education
2008-2013 PhD in Molecular Biotechnology, RWTH Aachen University,
Aachen, Germany.
2004-2006 Master of Philosophy (M. Phil) in Biotechnology and Genetic Engineering,
NWFP Agricultural University, Peshawar, Pakistan.
2000-2004 BSc (Hons) in Plant Breeding and Genetics, NWFP Agricultural
University, Peshawar, Pakistan.
1996-1999 Higher Secondary School Certificate, Peshawar, Pakistan.
1986-1996 High School Certificate, Peshawar, Pakistan.