Protein Engineering.pptx

BIOTECHNOLOGY IS CHALLENGING SUBJECT TO TEACH AND UNDERSTAND ALSO

1. Protein Engineering

2. Protein Engineering PROTEIN + ENGINEERING Combination of Designing and Amino acid developing Protein Engineering is the process of designing of new enzyme or protein with new or desirable function. Process of developing useful or valuable proteins. It is based on A)rDNA Technology B)Chemical Treatment

3. EX. Formation of protein depends upon two steps Transcription Translation TRANSCRIPTION TRANSLATION DNA RNA PROTEINS Process of formation of DNA to RNA is called Transcription . Process of formation of RNA to PROTEIN is called Translation .

4. In Genetic Engineering formation of DNA changes with r DNA technology to increase amount of protein. Ex. RE DNA Cut DNA New DNA cut by Restriction Enzyme New DNA

5. OBJECTIVE Production of proteins / Enzymes in large quantity To create a superior enzyme to catalyze the production of high specific chemical. Thermal and pH stability should be high. Eliminate the need of cofactor ( non protein molecules ). Changing of substrate binding site. Synthesizing enzyme should be stable and active in non –aqueous solvent. Make isolation and purification of enzyme simpler.

6. Methods of Protein Engineering Mutagenesis RDNA technology 1. Mutagenesis Change in DNA sequence is called mutagenesis DNA sequence changed by 3 steps 1.Substitution - change in one nucleotide. 2.Insertion – Gaining of additional nucleotide 3.Deletion- Deleting of one nucleotide . C C X A-A-A A—A A-A Substitution Insertion Deletion

7. Genetic Modification Modification of protein through genes is an easiest and more efficient approach compared to chemical modification. The basic method of genetic engineering is the modification of responsible gene and generate the proteins. Gene modification involves 2 process 1 . In vitro mutagenesis using synthetic oligonucleotide . 2 . Synthesis of complete modified gene.

8. PE METHOD DIRECT MUTAGENESIS RATIONAL PROTEIN DESIGN SITE DIRECTED MUTAGENESIS RANDOM MUTAGENESIS STUDY OF SEQUENCE AND STUDY OF PROTEIN STUCTURE. Mutagenesis on particular gene Mutation on all areas of gene THIS PROCESS IS INEXPENSIVE AND EASY

9. WORKING OF PE RATIONAL DESIGN APPROACH DIRCTED EVALUATION SITE DIRECTED MUTAGENSIS RANDOM MUTAGENESIS TRANSFORMATION TRANSFORMATION PROTEIN EXPRESSION PROTEIN EXPRESSION ISOLATION AND PURIFICATION OF PROTEIN ISOLATION AND PURIFICATION OF PROTEIN EVALUATION AND TESTING OF ACTIVE OR NEW ENZYME

10. CHEMICAL MODIFICATION Chemical modification has more importance in site directed mutagenesis. In this method functional group on side chain of natural enzyme may be changed or original protein modified & replaced. The protein modification is used for increase the stability of enzymes to high temperature & organic solvents. The amino acid lysine residues can be cross linked by use of chemical linkers. The most commonly used protein cross linker is glutaraldehyde . This method is only applicable to amino acids that have reactive side chains and surface proteins.

11. Ex. CHEMICAL MODIFICATION DNA TRANSCRIPTION RNA TRANSLATION PROTEIN POST TRANSLATION MODIFICATION BY CROSS LINKING USING CROSS LINKERS EX . GLUTARALDEHYDE

12. APPLICATION PHARMACEUTICAL OR MEDICAL APPLICATION- It is used to produce therapeutic pharmaceutical protein with high solubility & stability. It is used for production of biotechnology drugs & treatment of disease by vaccine , gene therapy. It is used in cancer treatment. Novel antibodies are used as anticancer agents. It is widely used in development of secreted protein such as insulin , interferon , erythropoietin as biotherapeutic agent.

13. 2.Food & Detergent Industry Wheat gluten protein is the one of the best ex. of protein engineering in food industry. Protease , Amylase & Lipase are important enzymes for Industrial , Food , Detergent Applications. Protease are commonly used in Food Industry as Flavors. Amylase is used for saccharification of starch ( the process of breaking a complex carbohydrate into monosaccharide components) & bread softness in food industry. It used in Detergent Industry for removal of starch strains. Lipases used for stability & cheese flavor .

14. 3.Environmental Application It is used for improvement in microbial strains. Oxygenase & Peroxides are used in treatment of organic pollutant. It is commonly used for modification of enzymes for waste management and pollution control.

15. 4.Other Application It is used to produce peptide based biomaerial such as elastin like polypeptides, silk like polymers etc. PE methods are used to improve physical stability of various particles. Virus particles are modified by Protein Engineering & used in medicine, as a new vaccines, gene therapy, targeted drug delivery vectors.

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