proteinengineering saurav 110510012515 phpapp02

Upload: rednri

Post on 05-Apr-2018

222 views

Category:

Documents


0 download

TRANSCRIPT

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    1/23

    SEMINAR ON

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    2/23

    PROTEINENGINEERING Protein engineering can be defined as

    the modification of protein structure withrecombinant DNA technology or

    chemical treatment to get a desirablefunction for better use in medicine,industry and agriculture.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    3/23

    OBJECTIVES OF PROTEINENGINEERING The objectives of protein engineering is as

    follows

    (a) to create a superior enzyme to

    catalyze the production of high valuespecific chemicals.

    (b) to produce enzyme in large quantities.

    (c) to produce biologicalcompounds(include synthetic peptide,storage protein, and synthetic drugs)superior to natural one.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    4/23

    RETIONALE OF PROTEINENGINEERING

    For industrial application an enzyme,should possess some characteristics inaddition to those of enzymes in cells.

    These characteristics are :-(1) enzyme should be robust with long

    life.

    (2) enzyme should be able to use thesubstrate supplied in the industry even itdiffers from that in the cell.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    5/23

    (3) enzyme should be able to workunder conditions, e.g. extreme of pH,temperature and concentration of the

    industry even if they differ from those inthe cell.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    6/23

    In view of above, the enzyme should beengineered to meet the altered needs.Therefore efforts have been made to

    alter the properties of enzymes. These are some character that one

    might have to change in a predictable

    manner in protein engineering orenzyme engineering to get the desiredfunction :-

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    7/23

    Kinetic properties of enzyme-turnover

    and Michaelis constant, Km. Thermo stability and the optimum

    temperature for the enzyme.

    Stability and activity of enzyme innonaqueous solvents.

    Substrate and reaction specificity.

    Cofactor requirements

    Optimum PH. Molecular weight and subunit structure.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    8/23

    Therefore for a particular class of

    enzymes, variation in nature may occurfor each of the above properties, so thatone may like to combine all the optimumproperties to the most efficient form of

    the enzyme.

    For an e.g. glucose isomerases, whichconvert glucose into other isomers like

    fructose and are used to make highfructose corn syrup vital for soft drinkindustries.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    9/23

    Basic assumption for protein engineering

    While doing protein engineering shouldrecognize the following properties ofenzymes, many amino acid substitution, deletions or

    additions lead to no changes in enzyme activityso that they are silent mutator.

    Protein have limited number of basic structuresand only minor changes are superimposed onthem leading to variation

    Similar patterns of chain folding and domainstructure can arise from different amino acidsequences with little or no homology.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    10/23

    Steps involved in proteinengineering

    A study of three dimensional structure ofprotein :-

    A study of three dimensional structure

    is the preliminary steps of proteinengineering. And a 3d structure ofprotein is produced from the data

    generated from X-ray crystallographyand NMR process by protein modeling.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    11/23

    The three-dimensional structure of penicillin, for which Dorothy Crowfoot Hodgkinwas awarded the Nobel Prize in Chemistry in 1964. The green, white, red, yellow andblue spheres represent atoms of carbon, hydrogen, oxygen, sulfur and nitrogen,respectively.

    http://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Dorothy_Crowfoot_Hodgkinhttp://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Sulfurhttp://en.wikipedia.org/wiki/Nitrogenhttp://en.wikipedia.org/wiki/Nitrogenhttp://en.wikipedia.org/wiki/Sulfurhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Hydrogenhttp://en.wikipedia.org/wiki/Carbonhttp://en.wikipedia.org/wiki/Nobel_Prize_in_Chemistryhttp://en.wikipedia.org/wiki/Dorothy_Crowfoot_Hodgkinhttp://en.wikipedia.org/wiki/Penicillinhttp://en.wikipedia.org/wiki/Image:Penicillin.png
  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    12/23

    Ribbon diagram of the structure of myoglobin determined with the x-raycrystallography

    http://en.wikipedia.org/wiki/Ribbon_diagramhttp://en.wikipedia.org/wiki/Myoglobinhttp://en.wikipedia.org/wiki/Myoglobinhttp://en.wikipedia.org/wiki/Ribbon_diagramhttp://en.wikipedia.org/wiki/Image:Myoglobin.png
  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    13/23

    Pacific Northwest National Laboratory's high magnetic field (800 MHz, 18.8 T) NMRspectrometer being loaded with a sample.

    http://en.wikipedia.org/wiki/Pacific_Northwest_National_Laboratoryhttp://en.wikipedia.org/wiki/Tesla_(unit)http://en.wikipedia.org/wiki/Tesla_(unit)http://en.wikipedia.org/wiki/Pacific_Northwest_National_Laboratoryhttp://en.wikipedia.org/wiki/Image:Pacific_Northwest_National_Laboratory_800_MHz_NMR_Spectrometer.jpg
  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    14/23

    Methods for protein engineering

    A variety of methods are used in proteinengineering such as mutagenesis,selection and recombinant DNA

    technology.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    15/23

    Mutagenesis

    Mutagenesis and selection can beeffectively utilized fro improving a specificproperty of an enzyme.

    E.g. for E.coli anthranilate synthetase

    enzyme is normally sensitive to tryptophaninhibitor due to feedback inhibition but analtered MTR2 mutation of E.coli was foundto possess an altered form of enzymeanthranilate synthetase that is insensitiveto tryptophan inhibition. And thus helping inthe continuous synthesis of tryptophanwithout inhibition.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    16/23

    Gene Modification

    The two process of gene modification are-

    (a) In vitro mutagenesis using syntheticoligonucleotides.

    (b) Synthesis of complete modified genede novo.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    17/23

    (a) In vitro mutagenesis using syntheticoligonucleotides.

    Synthetic oligonucleotides is used forinvitro mutagenesis. In this method a smalloligonucleotides primer containing the

    desired modification is first synthesized. Itis then hybridized to the appropriate siteand cloned gene and then the rest isreplicated using DNA polymerase enzyme,

    so that the rest remains unaltered. Thisapproach is actually used to modify theactive site of the tyrosyl-tRNA synthetase

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    18/23

    Synthesis of complete modified gene denovo.

    Complete gene in some cases havebeen chemically synthesized in the formof several oligomers (e.g. genes for

    insulin, somatostain and interferon), thatare ligated in correct order to produce acomplete gene. The sequence of thesynthetic gene can be designed in amodular fashion to get the desiredfunction.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    19/23

    Chemical modification ofenzymes

    The protein synthesized under the controlof gene sequence in a cell undergo post-transitional modification. This leads tostability, structural integrity, altered

    solubility and viscosity of individualproteins.for e.g. Enzyme-PEG conjugates. An

    enzyme L-asparaginase has antitumourproperties but is toxic with a life time of lessthen 18hrs thus reducing its utility. L-asparginase can be modified bypolyethene glycol derivatives to produce

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    20/23

    PEG-asparginase conjugates, which differfrom the native enzyme in the followingway (i) it retains only 52% of the

    catalytic activity of the native. (ii) itbecome resistant to proteolyticdegradation. (iii) it doesnt cause allergy.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    21/23

    Achievements of proteinengineering A number of proteins are known now

    where efforts have been made to knowthe effects of site specific mutagenesis

    involving substitution of one or moreamino acids.

    Insulin- it consist of A and B chainsare linked by C-peptide of 35 amino

    acids. It was shown that a sequence of 6amino acids for c-peptide was adequatefor the linking function.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    22/23

    cytochrome c A phenylalanine residuehas been identified to be non-essentialfor electron transfer but is involved in

    determining the reduction potential ofthe protein.

    Trypsin- It could be redesigned to have

    altered substrate specificity.

  • 7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02

    23/23