Protein Engineering
description
Transcript of Protein Engineering
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Prepared by :
Danda Pani ChapagainPramod NiraulaGopal KarkiMukesh MaharjanUjjwal BhushalPrem BhatBijaya Sharma
Submitted to:
Dr. Pramod Aryal(lecturer of Animal Biotechnology,SANN College, Gaihridhara)
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The design and construction of new proteins or enzymes with novel or desired functionsby modifying amino acid sequences by using recombinant deoxyribonucleic acidtechnology(RDT).
Protein engineering is the application of science, mathematics, and economics to the process of developing useful or valuable proteins. It is a young discipline, with much research currently taking place into the understanding of protein folding and protein recognition for protein design principles.
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The major assumptions of the method are:
(1) mutation does not significantly change thestructure of the folded state
(2) the target groups do not make newinteractions with new partners during thecourse of reaction energy
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DESIGNING BETTER PROTEIN
Thermodynamics
Folding
Biochemical
Structure
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Methods of protein engineering :
Site Directed Mutagenesis (rational design)
Random Mutagenesis (direct evolution)
DNA Shuffling
Fusion proteins
Glycosylation
PEGylation
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Detailed knowledge of the structure and function of the protein is used to make desired changes.
This has the advantage of being generally inexpensive and easy, since site‐directed mutagenesis techniques are well‐developed.
For the rational design we should have detailed knowledge of gene sequence and protein structure .
However, there is a major drawback in that detailed structural knowledge of a protein is often unavailable, and even when it is available, it can be extremely difficult to predict the effects of various mutations.
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• Computational protein design algorithms seek to identify amino acid sequences that have low energies for target structures.
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1) To alter a single amino acid residue by
mutating the codon that encodes for that amino acid.
ATG GCC GGA GAC GAG ACT ACT AAA
ATG GCC GGA GTC GAG ACT ACT AAA
translates to…..
Met ‐ Ala ‐ Gly ‐ Asp ‐ Glu ‐ Thr ‐ Thr ‐Lys
Met ‐ Ala ‐ Gly ‐ Val ‐ Glu ‐ Thr ‐ Thr ‐Lys
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Application
Medicine
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Example:
• Effectiveness of Ribonuclease (RNase) used in anti tumor therapy can be improved by this site directed mutagenesis.
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Engineered Dimeric human pancreatic RNase
Leu
Cys
Lys+
Cys
Leu
cys
Lys+
Cys
Gln
Arg
Asn
Arg
Dimerization
After Protein EngineeringBy site directed mutagenesis
Native Monomeric Human Pancreatic RNase
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2) To create a new restriction site for manipulation of DNA withoutintroducing an amino acid change.
AAT TCG CAT TCT ATG GGT ACC Asn‐ Ser ‐ His ‐ Ser ‐Met ‐ Gly ‐Thr
NcoI
AAT TCG CAT T(CC ATG G)GT ACCAsn‐ Ser ‐ His ‐ Ser ‐Met ‐ Gly –Thr
New restriction site, no change in amino acid sequence. TCT = Ser, TCC = Ser
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• Possible without knowledge about sequence/structure
• Generation of mutant libraries
• efficient screening
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Random mutagenesis is applied
selection regime is used (for protein)
variants
Further rounds of mutation and selection are then applied
produces superior results to rational design
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Error prone PCR:
• based on the principle that Taq polymerase is capable of annealing incompatible base‐pairs to each other during amplification under imperfect PCR conditions.
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• Error prone PCR:
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• 7 mMMgCl2• add H2O to 100 μl• 1 μl Taq• 2 μM Primer as• 2 μM Primer s• 100 ng Template• 0,2 mM dATP• 0,2 mM dGTP• 1 mM dCTP• 1 mM dTTP• 0,5 mMMnCl2• 20 mM Tris (pH 8.4)• 50 mM KCl
4°C72°C 10 min72°C 3 min58°C 45 s 30 x94°C95°C 3 min
Error prone PCR:
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restriction
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To mimic the natural design process and speed it up bydirected selection in vitro toward a simple specific goal.For example: protein engineering
In vitro homologous recombination of pools of selectedgenes by random fragmentation and polymerase chainreaction reassembly
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What do we need?
• applied to sequences > 1Kb
• Presence of homologous regions separating regions of Diversity
• Scaffold‐like protein structures may be particularly Suitable for shuffling
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Phylogenetic tree of 4 cephalosporinase genes
% sequencesimilarity
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Chimeric DNA sequences
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Alpha interferons (IFN‐s) are members of the diverse
helical‐bundle superfamily of cytokine genes.
• The human IFN‐s (Hu‐IFN‐s) are encoded by a family of over 20 tandemly duplicated nonallelic genes that share 85–98% sequence identity at the amino acid level.
• These proteins have potent antiviral and antiproliferative activities that have clinical utility as anticancer and antiviral therapeutics. Although the utility of chimeric IFNs derived from this gene family has been recognized, only a small fraction of the 1026 possible chimeras have been explored either in natural human evolution or by the methods of modern molecular biology; and only one natural IFN‐ subtype, Hu‐IFN‐2, has been used in clinical studies.
• The most active engineered IFN‐, IFN alfacon‐1, is a consensus of 13 wild‐type Hu‐IFN‐ genes that is currently used in hepatitis C therapy.
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• A novel protein engineered by fusing the protein coding sequence of one gene to the protein coding sequence of a different gene.
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Can be used to direct toxins to a target site.
(IL‐2 + Diptheria toxin)
aa 2nd ‐133 human IL‐2 + 1st ‐389 aa of diptheria toxintargets IL‐2 receptors on CTCLs to kill them
Denileukin diftitox (Ontak) ‐ FDA approved for cancer30% patients have 50% reduction in tumor burden
CTCL = cutaneous T‐cell lymphoma
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Ontak IL‐2 + diptheria toxin fusion protein
Ontak binds to surface of lymphoma cells via IL‐2
receptor
Once internalized, diptheria toxin kills cell.
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•Add/remove glycosylation sites
•Change biochemical feature/activity of the protein
•Improve stability
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Altering Glycosylation Sites :
• Use site‐directed mutagenesis to introduce new glycosylation sites.
• Erythropoietin as an exampledirect relationship between carbohydrate content (sialic acid) and its serum half life and biological activity in vivo
Inverse relationship with receptor binding (i.e., more glycosylation means poorer binding)
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• Hypothesis was:the more glycosylation ‐‐> longer half life
‐‐> more activity‐‐> reduced binding affinity
• Hyperglycosylated EPO (Aranesp)
• In vivono loss of drug functionincreased serum half‐life (3‐fold longer)reduced frequency of administration
• FDA approved 1/30/03 for anemia
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• Similarly, PEGylation (monomethoxypolyethylene glycol ) is also in use to improve the protein stability and activity.
• Protein drugs have:
relatively short half‐lives
wide tissue distribution
potential for immunogenicity
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• It is used to design the novel protein that has the enhanced activity.
• Protein engineering is the combination of science, mathematics and management.
• It can be applied in the fields like‐medicine, industries,agriculture etc…
• Since it is very tough task, many researches is still done.
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• Glick.B,R, Pasternark,j,j, Molecular Biotechnology, Principles in Applications ofRecombinant DNA,IIIrd edition ,ASM press washington D.C.
• Fresht,A. Structure and Mechanism in protein science,w.h. freeman and company,New York.
• Chawala,H.S. Introduction to Plant Biotechnology, IInd edition, Oxford and IBHpublishing Co.Pvt. Ltd.,New Dehli.
• http://www.w3.org
• http://nar.oxfordjournals.org
• http://ncbi.nlm.nih.gov/pubmed
• http://www.upei.ca