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SEMINAR ON
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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.
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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.
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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.
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(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.
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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 :-
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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.
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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.
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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.
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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.
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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.png7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02
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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.png7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02
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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.jpg7/31/2019 Proteinengineering Saurav 110510012515 Phpapp02
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Methods for protein engineering
A variety of methods are used in proteinengineering such as mutagenesis,selection and recombinant DNA
technology.
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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.
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Gene Modification
The two process of gene modification are-
(a) In vitro mutagenesis using syntheticoligonucleotides.
(b) Synthesis of complete modified genede novo.
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(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
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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.
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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
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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.
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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.
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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.
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