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9 Vuorela, A., Myllyhaju, J. Nissi,
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I 263- I2 70
15 Fertala, A., Ho lmes , D.F., Kadler, K. E., Sieron, A. L. and
Prockop, D.J. ( 1996) J Biol. Chem. 27 I , 14864- I48 69 Received
28
February
2000
Expression of recombinant human type 1-111 collagens in the yeast Pichia pastoris
J. Myllyharju ,M.Nokelainen, A. Vuorela and K.
1
Kivirikko
Collagen Research Unit, Biocenter and Department of Medical B iochemistry, University
of
Oulu, P.0 Box 5000,
FIN-900
I 4 Oulu, Finland
Abstract
An efficient expression system for recombinant
human collagens will have numerous scientific
and medical applications. However, most recom-
binant systems are unsuitable for this purpose, as
they do not have sufficient prolyl 4-hydroxylase
activity. We have developed methods for pro-
ducing the three major fibril-forming human
collagens, types I, I1 and 111, in the methyl-
otroph ic yeast
Pichia
pastoris.
These methods are
based on co-expression of procollagen polypeptide
chains with the a and P-subunits of prolyl 4-
hydroxylase. T h e triple-helical type-I,
-11 and
-111 procollagens were found to accumulate pre-
dominantly within the endoplasmic reticulum of
the yeast cells and could be purified fro m the cell
lysates by a procedure that included a pepsin treat-
ment to convert the procollagens into collagens
and to digest most of the non-collagenous proteins.
All the purified recombinant collagens were ident-
ical in 4-hydroxyproline conten t with t he corres-
ponding non-recombinant human proteins, and
all the recombinant collagens formed native-type
fibrils. T h e expression levels using single-copy
integrants and a
2
litre bioreactor ranged from
0.2
to
0.6
g/l depending on the collagen type.
Key words: methylotrophic yeast, procollagen, prolyl 4-hy-
droxylase.
Abbreviation s used: aMF,a matingfactor; proa
I (I),
proa 11) and
proa
I (Ill) chains, proa I chains
of
type-I,
11
and -111 procollagen,
respectively; proa2(1) chain, proa2 chain of type-I procollagen.
To
whom correspondence should be addressed (e-mail
Introduction
The collagen family consists of about
20
proteins
formally defined as collagens and more than 10
additional proteins with collagen-like domains
[l-31.
Type-I collagen is now used as a biomaterial
in numerous medical applications and as a delivery
system for various drugs [4-61. In addition, all
gelatins are made from collagens. Th e collagens
used in all these applications have been isolated
from animal tissues and are liable to cause allergic
reactions in some subjects and carry a risk of
disease-causing contaminants. Th e various colla-
gen types have different properties, and therefore
some of the other collagens might be more suitable
for certain applications than type I. However, it
has been difficult or impossible to isolate sufficient
quantities of the other collagens from animal
tissues. It is obvious, therefore, that an efficient
large-scale recombinant system for the production
of human collagens would have numerous ap-
plications in medicine.
Most recombinant systems now available for
large-scale production of proteins cannot be used
as such for the production of recombinant colla-
gens, as bacteria and yeast have no prolyl 4-
hydroxylase activity, and insect cells [7] and the
mammary gland [8] have insufficient levels of th is
enzyme activity. Prolyl 4-hydroxylase, an aJI
tetramer in vertebrates, plays a central role in the
synthesis of all collagens, as the 4-hydroxyproline
residues formed are essential for the folding of the
newly synthesized collagen polypeptide chains
into triple-helical collagen molecules [2 9 10].
353
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Biochemical Society Transactions (2000) Volume 28, part 4
Therefore, the recombinant collagen polypeptide
chains expressed in most systems will remain as a
non-triple-helical, non-functional protein or, if
the cells are grown at low temperatures, the chains
may form molecules with unstable triple helices.
We have demonstrated that co-expression of
polypeptide chains of various types of human
collagen with the two types of subunit of human
prolyl 4-hydroxylase can be used for efficient
recombinant expression of human collagens in
insect cells [7,11,12]. The recombinant type 1-111
collagens produced have been very similar if not
identical with the corresponding non-recombinant
proteins in their 4-hydroxyproline contents and
various other properties, and the highest ex-
pression levels obtained in suspension cultures
have ranged up to about 50mg/l [7,11,12]. In
addition, it has been demonstrated that this same
principle can be used for the high-level production
of an engineered form of human type-I collagen in
mouse milk [8]. We have recently applied the
principle to the high-level production of recom-
binant human type-1-111 collagens in the methyl-
otrophic yeast Pichia pastoris.
Expression of an active recombinant
human prolyl4-hydroxylase tet ram er
and the effect of co-expression with
collagen polypeptide chains
In order to study whether subunits of human
prolyl 4-hydroxylase are able to form an active
enzyme tetramer in yeast cells, cDNAs for the
human a and 8-subunits were cloned into the
Pichia expression vectors pARG815 (comple-
menting for arg4 in the host) and PA0815 (com-
plementing for his4 in the host), respectively, and
co-transformed into the GS200 (his4, arg4) P .
pastoris host train [13]. Initial attempts to express
an active human prolyl4-hydroxylase tetramer in
P. pastoris were only partially successful, as only a
minor fraction of the recombinant polypeptides
expressed were found in the form of the tetramer,
whereas the vast majority were present in un-
assembled forms [13]. A much higher tetramer
assembly level was obtained [13] when the signal
peptide of the /?-subunit was replaced by the
Saccharomyces cerevisiae a mating factor
(aMF)
pre-pro sequence by cloning the P-subunit c DNA
into the expression vector pPIC9 (generating
vector pPIC9p). This signal sequence gave the
highest amount of tetramer among the various
constructs studied, even though it also markedly
increased the secretion of the P-subunit into the
culture medium. Even in this P. pastoris strain,
however, the vast majority of the a andp-subunits
were found in unassembled forms.
T o study the expression of recombinant
human collagens in P. pastoris, cDNAs for the
proal chains of type-I, -11 and -111 procollagens
[proal(I), proal(I1 ) and proal (III)] were cloned
separately into the expression vector pPICZB and
transformed into a recombinant P. pastoris strain
expressing human prolyl 4-hydroxylase subunits
in which the 8-subunit had the S. cerevisiae a M F
pre-pro sequence ([13,14] and M. Nokelainen, A.
Vuorela, K. I. Kivirikko and J. Myllyharju, un-
published work). A highly unexpected finding was
that co-expression of the prolyl 4-hydroxylase
subunits with any of these procollagen polypep-
tide chains led to an up-to-about- 10-fold increase
in the amount of the enzyme tetramer with no
increase in the total amounts of its subunits
([13,14] and
M.
Nokelainen, A. Vuorela, K. I.
Kivirikko and J. Myllyharju, unpublished work).
Pulse-chase experiments indicated that the half-
lives of the recombinant enzyme tetramers ex-
pressed in
P.
pastoris without co-expression with
collagen polypeptide chains were only about
50 min [14], while co-expression with the proa-
l( II1) chains increased this half-life to 12.5 h and
co-expression with the p roal ( I) chains gave a half-
life of 6.5 h, i.e. 8 times that of the strain expressing
the enzyme alone but
50%
of that of the strain
co-expressing prolyl 4-hydroxylase with the
proal (I1 ) chains [141. The difference in half-life
between the strains co-expressing the proal (I )
and proal( II1) chains is likely to be related to the
level of procollagen expression, that of type-I
procollagen homotrimers being 35-70 yoof that of
type-I11 procollagen. T he data thus indicate that
collagen synthesis in P .pastoris, and probably also
in other cell types, involves a highly unusual
control mechanism, in that the production of a
stable prolyl 4-hydroxylase tetramer requires the
expression of collagen polypeptide chains, whereas
the production of collagen molecules with stable
triple helices requires the expression of active
prolyl 4-hydroxylase [13,141.
Expression of human type-I -11 and -111
collagens in shaker flasks
The strains described above were used to study
the expression of recombinant type-I , -11 and -111
procollagen homotrimers in P. pastoris. In order
to express type-I procollagen heterotrimers, a
cDNA for the proa2 chain of human type-I
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of
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procollagen [proa2(1)] was cloned into the
pBLA DE IX vector (complementing for adel in
the host; M. Nokelainen, H. Tu, A. Vuorela,
H. Notbohm , K. I. Kivirikko and J. Myllyharju,
unpubl ished work). A stra in expressing prolyl 4-
hydroxylase was first generated to a yJC300 (his4,
arg4, adel)
P.
pastoris host strain by cloning a
cDNA for the a-subunit into the pBLARG IX
vector (complementing for arg4 in the host), and
this construct was co-transformed with the
pPIC9/3 expression vector (see above) into the
yJC300. This was followed by subsequent trans-
formations of the pPICZB vector containing the
proal(1) cDNA and pBLADE IX vector con-
taining th e proa2( I) cD NA.
All the P. pastoris strains expressing pro-
collagen were found
to
produce full-length proa
chains ([13,14] and M. Nokelainen, A. Vuorela,
K. I. Kivirikko and J . Myllyharju, unpublished
work). Th e p r o d (I) chains, when expressed
alone, and the proal(I1) and proal(II1) chains,
each formed triple-helical molecules with collagen
domains that were resistant
to
pepsin digestion,
whereas no pepsin-resistant chains were obtained
when the proa2(1) chains were expressed alone.
Co-expression of the proal(1 ) and proa2(I) chains
led to the formation of heterotrimeric type-I
procollagen molecules with the correct 2
:
1 chain
ratio (M. Nokelainen, H. Tu, A. Vuorela,
H.
Notbohm, K. I. Kivirikko and J. Myllyharju,
unpublished work). Studies by SDS/PAGE
under reducing and non-reducing conditions
indicated that all the proa chains and also the
a1 111) chains produced by pepsin digestion
from the corresponding procollagen molecules
formed disulphide-bonded trimers ([13] and M.
Nokelainen,
A
Vuorela,
K .
I. Kivirikko and J.
Myllyharju, unpublished work).
T h e thermal stability of the pepsin-resistant
recombinant collagens was studied using digestion
with a mixture of trypsin and chymotrypsin after
heating to various temperatures
[15].
T h e
T
values of the recombinant type 1-111 collagens
were approx. 38 C, which is 2-3
C
lower than
that found in vivo ([13] and J. Myllyharju, M.
Nokelainen, A. Vuorela and K.
I .
Kivirikko, un-
published work). Amino acid analysis
of
the
recombinant type-I I I collagen purified from
shaker-flask cul tures showed that the degree of 4-
hydroxylation
of
the proline residues was 44.2
yo
whereas the corresponding value for non-recom-
binant human type-I I I collagen was
5
1.6
[
131.
The best level
of
type-I11 collagen expression
obtained in shaker-flask cultures was approx.
15 mg/l, whereas the levels obtained for type-I
and -11 collagens were approx. 35-70
yo
of that of
type-I11 collagen ([13,14] and M. Nokelainen,
A. Vuorela, K. I. Kivirikko and J. Myllyharju,
unpublished work).
The triple-helical type-I,
-11
and
-111
pro-
collagen molecules produced in
P.
pastoris were
found to accumulate predominantly inside the
yeast cell, only about 10% being found in the
culture medium. This is surprising, as triple-
helical procollagen molecules are rapidly secreted
into th e extracellular space from various animal
cells. Replacement of the signal sequence of the
human proal(II1) chain with the
S.
cerevisiae
a M F pre-pro sequence led
to
only a slight im-
provement in secretion, and the total expression
level
of
type-I11 procollagen with the a M F
pre-pro sequence was lower than that with the
authentic signal peptide [16]. Immunoelectron
microscopy indicated that the recombinant pro-
collagen molecules accumulated within th e endo-
plasmic reticulum and did not proceed any further
in the secretory pathway [16]. Th e lackof secretion
may have been related to the large size
of
the
procollagen molecule.
Expression of human type-I -11 and -111
collagens in
a
bioreactor
Many previous studies have shown that shaker-
flask conditions are not optimal for protein pro-
duction in P. pastoris, du e to the lack of sufficient
0
and marked increases in expression levels are
usually obtained in bioreactors
[
17,181. As the
K
of
0
in the prolyl4-hydroxylase reaction is about
40 pM [19], the
0
concentration within the lumen
of the endoplasmic reticulum is also likely to be
rate-limiting for hydroxylation in shaker-flask
cultures. Thus it could be expected that the
differences in 4-hydroxyproline conten t between
the recombinant and non-recombinant collagens
may disappear when the recombinant collagens
are produced in a bioreactor. The type-I pro-
collagen homotrimers and heterotrimers and the
type-I1 and -111 procollagens were therefore
expressed in a 2 litre B. Braun Biostat
C
bioreactor
equipped with an
0
supply system, whereupon
their expression levels were indeed markedly
higher than in the shaker-flask cultures, ranging
from about 0.2
to 0.6
g/1 (M . Nokelainen,
H.
T u ,
A.
Vuorela, H. Notbohm, K. I. Kivirikko and J.
Myllyharju, unpublished work). It should be
noted that all the experiments reported in this
paper were carried out with single-copy integ-
rants. It has been demonstrated previously that
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Biochemical Society Transactions (2000) Volume 28, part 4
the levels of expression of various proteins in
P.
pastoris increase markedly with the number of
DN A copies, at least up to
30 50
copies [17,20,21].
Th e present system can thus be optimized for the
production of very large amounts of various
collagens.
The recombinant collagens produced in the
bioreactor were purified by pepsin digestion and
selective salt precipitation followed by Sephacryl
S-500HR
gel filtration in the AKTA explorer
system (Amersham Pharmacia Biotech). All the
recombinant collagens produced were found to be
essentially pure when analysed by SDS/PAGE
followed by Coomassie Brilliant Blue staining
(Figure1).Amino acid analyses showed that the
4
hydroxyproline contents of all the purified recom-
binant collagens were identical with those reported
for the corresponding non-recombinant human
proteins (M. Nokelainen, A. Vuorela, K . I . Kivi-
Figure I
SDSlPAGE
analysis
of
purified recombinant human
collagens expressed in P.
pastoris
The long arrow indicates the a1 chains of the type-I collagen
homotrimer (lane I ) and heterotrimer lane2), and type-ll (lane3)
and type-Ill collagens (lane 4). The short amw indicates the a2
chain of the type-I collagen heterotrimer (lane
2).
1 2 3 4
Figure
2
N-termini
of
the a-chains of the purified recombinant
human collagens
The N elopeptide sequences are underlined. The amws indicate
the pepsin cleavage sites, while the N-terminal amino acid of the
a-chains of the final recombinant collagens is shown in bold.
J
a
1
I). . G N F ~ G G I S V P G P M G P S .
.
V W MGLM
...
2 I).. GNFMQYDGKG
a1@I ...NFM-GGAO hXMQGPMGPM ...
a1
@I ...Q
NYSPQYDSYDVKSGVAVGCL.AGYP
J
4
rikko and J. Myllyharju, unpublished work). N-
terminal sequencing of the polypeptide chains of
the recombinant collagens showed that in most
cases pepsin digestion had removed several resi-
dues from the N-terminus of the telopeptide
domain, but in the case of the
a2 I )
chain only one
residue had been removed and occasionally, if
pepsin digestion was incomplete, the
a2 I ) chains
had two additional N-terminal amino acids (i.e.
the cleavage had left the last two amino acids of the
N-propeptide on the N-terminus of the chain;
Figure 2). All the recombinant collagens produced
in P. pastoris were found to form native-type
fibrils (M. Nokelainen, H. Tu, A. Vuorela, H.
Notbohm, K.
I.
Kivirikko and J. Myllyharju,
unpublished work), which indicates that the dif-
ferences at the N-terminus do not influence the
fibrillar properties. This conclusion is supported
by a recent study on pepsin and pronase treatment
of rat non-recombinant type-I collagen molecules,
indicating that chains with shortened N-termini
form fibrils that are identical with those formed
from full-length chains [22]. It thus seems likely
that the recombinant procollagens produced in
P.
pastoris can be used for numerous applications
that currently require collagens purified from
animal tissues.
Wethank Dr.James Cregg, Keck Graduate nstitute of Applied Life
Sciences, for the gift of the P pastoris host strains and the
pBLARG IX and pBLADE IX vectors, and Raija Juntunen, Anne
Kokko, Eeva Lehtimaki, Minna Siunra and Tanja VaisLnen for their
expert technical assistance. This work was supported by grants
from the Health Sciences Council ofthe Academy of Finland, from
the European Commission B104-Cr96-0537),rom the National
Institutes of Health (ROI AR45879) and from FibroGen (South
San Francisco,CA, U.S.A.).
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Received I March
2000
Towards a fibrous compos ite with dynamically controlled stiffness
:
essons
from echinoderms
J. A. Tro t te r * , J. Tipper*, G. Lyons-Levy*, K.
Chino*,
A. H. Heuert . Z. L iu t ,
M.
Mrksichf,
C.
Hodnelandl,
W.
S.
Dillrnoref. T. J Koobtj, M. M. Koob-Ernundstj, K. Kadlery and D. Holrnesy
*Dep artment o f Cell Biology and Physiology, University of Ne w Mexico School
of
Medicine, Albuquerque,
NM 87 3 I, U.S.A., +Departme nt o f Materials Science and Engineering, Case Weste rn Reserve University,
I0900 Euclid Ave., Cleveland,
OH 44 106,
U.S.A.,
f
Department of Chemistry, Universrty of Chicago,
5735 S. Ellis Ave., Chicago, L 60637, U.S.A., SShrinen Hosp ital for Ch ildren, I2502 N. Pine Drive, Tampa,
FL 336 12, U.S.A., and flWellcome Trust Centre for Cell-Matrix Research, School of Biological Sciences,
unive rsity of Manchester, Stopford Building, Ox for d Road, Manchester M I 3 9PT,
U.K.
Abstract
Sea urchins and sea cucumbers, like other echino-
derms, control the tensile properties of their
connective tissues by regulating stress transfer
between collagen fibrils. The collagen fibrils are
spindle-shaped and up to 1 mm long with a
constant aspect ratio of approx.
2000.
They are
organized into a tissue by an elastomeric network
of fibrillin microfibrils. Interactions between the
fibrils are regulated by soluble macromolecules
that are secreted by local, neurally controlled,
effector cells. We are characterizing the non-linear
viscoelastic properties of sea cucumber dermis
under different conditions, as well as the struc-
tures, molecules and molecular interactions that
determine its properties. In addition, we are
developing reagents that will bind covalently to
fibril surfaces and reversibly form cross-links with
other reagents, resulting in a chemically controlled
stress-transfer capacity. The information being
developed will lead to the design and construction
of a synthetic analogue composed of fibres in an
Key words : collagen, interfibril lar cross-links, fibrils.
To whom correspondence should be addressed (e-mail:
elastomeric matrix that contains photo- or electro-
sensitive reagents that reversibly form interfib-
rillar cross-links.
Collagenous tissues
The structural materials of animals are, for the
most part, composites containing insoluble fibres
in a non-fibrous matrix. Familiar examples
of
such
materials include the tendons, ligaments and
dermis of mammals. Th e mechanical properties of
these fibrous composites are due largely to the
contributions of the protein collagen, which self-
assembles into long, thin fibrils that may be
millimetres in length and nanometres in diameter
[l] Collagen molecules (approx. 300 nm long x
1 5 nm in diameter) within the same fibril become
covalently cross-linked through enzymic action.
As a result
of
cross-linking, the fibrils possess
high tensile stiffness and strength (on the order
of GPa). I n most cases we
do
not know how long
the individual collagen fibrils are; nor do we
know how stress is transferred between them. We
do know, however, that the composition and
organization
of
the tissues is such as to make
effective use of the tensile properties of the fibrils.
In addition to collagen fibrils, connective tissues
357 2000
Biochemical Society