Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
Objective:
To create a superior enzymes to catalyze the production of high value specific chemicals.
To produce enzyme in large quantities.
Eliminate the need for co factor in enzymatic reaction.
Change substrate binding sites to increase specificity.
Change the thermal tolerance and pH stability.
Increase protein resistance to proteases.
To produce biological compounds.
Investigate how desired mutations can be introduced into a cloned gene
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
Objective:
To create a superior enzymes to catalyze the production of high value specific chemicals.
To produce enzyme in large quantities.
Eliminate the need for co factor in enzymatic reaction.
Change substrate binding sites to increase specificity.
Change the thermal tolerance and pH stability.
Increase protein resistance to proteases.
To produce biological compounds.
Investigate how desired mutations can be introduced into a cloned gene
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
UNIT-5 Protein Engineering: Brief introduction to protein engineering,Use of ...Shyam Bass
UNIT-5 6th Sem B.PHARMA PHARMACEUTICAL BIOTECHNOLOGY)
Protein Engineering: Brief introduction to protein engineering, Use of microbes in industry, Production of enzymes-general considerations, Amylase, Catalase, peroxidase, Lipase Basic principles of genetic engineering
BY- SHYAM BASS
Biosensors, Types of Biosensors, Applications of Biosensors, Nanotechnology, Nanobiosensors, Components of Biosensor, Working of Biosensor, Principle of Biosensor, Examples of Biosensor, Advantages of Biosensor, Disadvantages of Biosensor, Limitations of Biosensor, Features of a Biosensor, Calorimetric Biosensors, Potentiometric Biosensors, Acoustic Wave Biosensors, Amperometric Biosensors, Optical Biosensors, Examples of a Nanobiosensor, Lab on a chip,
Applications of Lab on a chip, Glucose Biosensor
Use of microbes in industry. Production of enzymes-General consideration-Amyl...Steffi Thomas
Industrial uses of microbes, properties of useful industrial microbes, various industrial products, production of enzymes-general consideration-amylase, catalase, peroxidase, lipase, protease, penicillinase, procedure for culturing bacteria and inoculum preparation, submerged fermentation and solid state fermentation, uses of different enzymes
The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!!
Mayur D. Chauhan
Basic principles of genetic engineeringSteffi Thomas
Basic principles of genetic engineering, Recombinant DNA, Genetically Modified organism (GMO), Tools used in genetic engineering, restriction endonuclease, DNA ligase, cloning vector, process of genetic engineering, applications of genetic engineering (in animals, plants, human), production of insulin by rDNA technology, gene therapy, possible hazards of genetic engineering
UNIT-5 Protein Engineering: Brief introduction to protein engineering,Use of ...Shyam Bass
UNIT-5 6th Sem B.PHARMA PHARMACEUTICAL BIOTECHNOLOGY)
Protein Engineering: Brief introduction to protein engineering, Use of microbes in industry, Production of enzymes-general considerations, Amylase, Catalase, peroxidase, Lipase Basic principles of genetic engineering
BY- SHYAM BASS
Biosensors, Types of Biosensors, Applications of Biosensors, Nanotechnology, Nanobiosensors, Components of Biosensor, Working of Biosensor, Principle of Biosensor, Examples of Biosensor, Advantages of Biosensor, Disadvantages of Biosensor, Limitations of Biosensor, Features of a Biosensor, Calorimetric Biosensors, Potentiometric Biosensors, Acoustic Wave Biosensors, Amperometric Biosensors, Optical Biosensors, Examples of a Nanobiosensor, Lab on a chip,
Applications of Lab on a chip, Glucose Biosensor
Use of microbes in industry. Production of enzymes-General consideration-Amyl...Steffi Thomas
Industrial uses of microbes, properties of useful industrial microbes, various industrial products, production of enzymes-general consideration-amylase, catalase, peroxidase, lipase, protease, penicillinase, procedure for culturing bacteria and inoculum preparation, submerged fermentation and solid state fermentation, uses of different enzymes
The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!!
Mayur D. Chauhan
Basic principles of genetic engineeringSteffi Thomas
Basic principles of genetic engineering, Recombinant DNA, Genetically Modified organism (GMO), Tools used in genetic engineering, restriction endonuclease, DNA ligase, cloning vector, process of genetic engineering, applications of genetic engineering (in animals, plants, human), production of insulin by rDNA technology, gene therapy, possible hazards of genetic engineering
Laboratory method for measuring enzyme activity.
Vital for study of enzyme kinetics and enzyme inhibition.
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Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
Protein engineering is the process of developing useful or valuable proteins.
Protein Engineering is a second generation of recombinant DNA technology.
It involves altering cloned DNA in vitro by novel mutational technique so that translated proteins have slightly altered properties.
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New development in herbals,
Bio-prospecting tools for drug discovery,
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2. PROTEIN ENGINEERING Protein engineering can be defined as the modification of protein structure with recombinant DNA technology or chemical treatment to get a desirable function for better use in medicine, industry and agriculture.
3. OBJECTIVES OF PROTEIN ENGINEERING The objectives of protein engineering is as follows – (a) to create a superior enzyme to catalyze the production of high value specific chemicals. (b) to produce enzyme in large quantities. (c) to produce biological compounds(include synthetic peptide, storage protein, and synthetic drugs) superior to natural one.
4. RETIONALE OF PROTEIN ENGINEERING For industrial application an enzyme, should possess some characteristics in addition to those of enzymes in cells. These characteristics are :- (1) enzyme should be robust with long life. (2) enzyme should be able to use the substrate supplied in the industry even it differs from that in the cell.
5. (3) enzyme should be able to work under conditions, e.g. extreme of pH, temperature and concentration of the industry even if they differ from those in the cell.
6. In view of above, the enzyme should be engineered 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 or enzyme engineering to get the desired function :-
7. Kinetic properties of enzyme-turnover and Michaelis constant, Km. Thermo stability and the optimum temperature for the enzyme. Stability and activity of enzyme in nonaqueous solvents. Substrate and reaction specificity. Cofactor requirements Optimum PH. Molecular weight and subunit structure.
8. Therefore for a particular class of enzymes, variation in nature may occur for each of the above properties, so that one may like to combine all the optimum properties to the most efficient form of the enzyme. For an e.g. glucose isomerases, which convert glucose into other isomers like fructose and are used to make high fructose corn syrup vital for soft drink industries.
9. Basic assumption for protein engineering While doing protein engineering should recognize the following properties of enzymes, many amino acid substitution, deletions or additions lead to no changes in enzyme activity so that they are silent mutator. Protein have limited number of basic structures and only minor changes are superimposed on them leading to variation Similar patterns of chain folding and domain structure can arise from different amino acid sequences with little or no homology.
10. Steps involved in protein engineering A study of three dimensional structure of protein :- A study of three dimensional structure is the preliminary steps of protein engineering. And a 3d structure of protein is produced from the data generated from X-ray crystallography and NMR process by protein modeling.
11. The three-dimensional structure of penicillin, for which Dorothy Crowfoot Hodgkin was awarded the Nobel Prize in Chemistry in 1964. The green, white, red, yellow and blue spheres represent atoms of carbon, hydrogen, oxygen, sulfur and nitrogen, respectively.
12. Ribbon diagram of the structure of myoglobin determined with the x-ray crystallography
13. Pacific Northwest National Laboratory's high magnetic field (800 MHz, 18.8 T) NMR spectrometer being loaded with a sample.
14. Methods for protein engineering A variety of methods are used inprotein engineering such as mutagenesis, selection and recombinant DNA technology.
15. Mutagenesis Mutagenesis and selection can be effectively utilized fro improving a specific property of an enzyme. E.g. for E.coli anthranilate synthetase enzyme is normally sensitive to tryptophan inhibitor due to feedback inhibition but an altered MTR2 mutation of E.coli was found to possess an altered form of enzyme anthranilate synthetase that is insensitive to tryptophan inhibition. And thus helping in the continuous synthesis of tryptophan without inhibition.
16. Gene Modification The two process of gene modification are- In vitro mutagenesis using synthetic oligonucleotides. Synthesis of complete modified gene de novo.
17. (a) In vitro mutagenesis using synthetic oligonucleotides. Synthetic oligonucleotides is used for invitro mutagenesis. In this method a small oligonucleotides primer containing the desired modification is first synthesized. It is then hybridized to the appropriate site and cloned gene and then the rest is replicated using DNA polymerase enzyme, so that the rest remains unaltered. This approach is actually used to modify the active site of the tyrosyl-tRNA synthetase
18. Synthesis of complete modified gene de novo. Complete gene in some cases have been chemically synthesized in the form of several oligomers (e.g. genes for insulin, somatostain and interferon), that are ligated in correct order to produce a complete gene. The sequence of the synthetic gene can be designed in a modular fashion to get the desired function.
19. Chemical modification of enzymes The protein synthesized under the control of gene sequence in a cell undergo post-transitional modification. This leads to stability, structural integrity, altered solubility and viscosity of individual proteins. for e.g. Enzyme-PEG conjugates. An enzyme L-asparaginase has antitumour properties but is toxic with a life time of less then 18hrs thus reducing its utility. L-asparginase can be modified by polyethene glycol derivatives to produce
20. PEG-asparginase conjugates, which differ from the native enzyme in the following way (i) it retains only 52% of the catalytic activity of the native. (ii) it become resistant to proteolytic degradation. (iii) it doesn’t cause allergy.
21. Achievements of protein engineering A number of proteins are known now where efforts have been made to know the effects of site specific mutagenesis involving substitution of one or more amino acids. Insulin- it consist of A and B chains are linked by C-peptide of 35 amino acids. It was shown that a sequence of 6 amino acids for c-peptide was adequate for the linking function.
22. cytochrome c – A phenylalanine residue has been identified to be non-essential for electron transfer but is involved in determining the reduction potential of the protein. Trypsin- It could be redesigned to have altered substrate specificity.