4 . Brief introduction to protein engineering.pptx

To give knowledge about topic.

1. Brief introduction to protein engineering 1 Prepared by: Ms. Harshada R. Bafna. M. Pharm (Quality Assurances)

2. Introduction 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 or in pharmaceuticals and other application. Protein engineering is the process of developing useful or industrial important proteins. Protein engineering involves synthesis of new proteins or to make changes in the existing protein sequence or structure to achieve desired function . 2

3. Fig 4.1: Basic principle of protein engineering Definition: 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

4. To create a superior enzyme. To develop more stable and catalytic efficient enzymes . To produce biological compounds (include synthetic peptide, storage protein, and synthetic drugs) superior to natural one . Get humanised antibodies with less immunogenicity . Get more site specific, more potent biopharmaceutical with altered pharmacological action. Over all aim of protein engineering application is to get functionally more useful enzymes, antibodies, hormones, receptor proteins. Change the substrate binding sites to increase specificity . Change the thermal tolerance and pH stability. objectives 4

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6. 1. Rational design approach Techniques of protein engineering It involve ‘site-directed mutagenesis’ of protein. Rational design play main role in protein engineering and manipulate different processes such as regenerative medicines, protein delivery system, tissue engineering etc. It improved by modification in the site directed mutagenesis technique , protein-protein interaction and protein 2D, 3D structure modeling . 6

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8. 2. Site-directed mutagenesis Site-directed mutagenesis is ‘in-vitro’ technique which allow introduction of specific amino acid into a target gene . It involve the change of cloned target DNA either by deletion, substitution or insertion of same host cell . This host cells are used for production of functional protein . The single-prime method is simplest method of site directed mutagenesis. It involve ‘ in-vitro’ DNA synthesis with chemically synthesized oligonucleotide (7 to 20 nucleotide) that carries a base mismatch with the complementary sequence. 8

9. A single stranded clone s of the wild type gene is produced by using M13 phage based vectors . The hybrid clone as a primer in the presence of DNA polymerase , synthesizes second DNA strand . The slightly mismatched duplex recombinant plasmid is used to transform bacteria. The duplex DNA replicates in bacterial cell and produce either wild type or mutant plasmid (Show in fig.4.3). 9

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11. 3. Chemical modification Chemical modification is most widely used and had more important before advances in site directed mutagenesis. In this method, f unctional group on side chain of natural enzymes may be changed or part of original protein modified and replaced . Protein modification is used for increase the stability of enzyme to high temp. and organic solvents. 11

12. Increasing the stability and biological activity of proteins The industrial application or therapeutic uses of enzymes/proteins can be appropriately brought into use by increasing their half-lives or thermostability. Proteins with enhanced stability can be obtained by following methods: Addition of disulfide bonds Changing asparagine to other amino groups Single amino acid changes Improving kinetic properties of enzymes Reducing the free sulfhydryl group 12

13. 1. Addition of disulfide bond Introduction of disulfide bonds significantly increases the thermostability of enzymes. The disulfide bonds added should not disturb the normal functioning of enzymes . The new protein obtained after the addition of disulfide bond does not unfold at high temperatures and also does not denatures at non physiological conditions (i.e high pH and presence of organic solvents). Eg: T4 lysozyme, xylanase 13

14. 2. Changing Asparagine to other amino acids The amino acids Asparagine and glutamine undergo deamination (i.e., release ammonia) to form aspartic and glutamic acid , respectively at high temperature . These alterations are associated with changes in protein folding and loss biological activity . Triose Phosphate isomerase- This is a dimeric enzyme with identical subunits , each having two thermosennsitive asparagine reduces which undergo deamination. 3. Reducing the free sulfhydryl group The presence of a large number of free sulfahydryl groups (contributed by cysteine residue) may lower the activity of proteins In case the stability and activity of the protein or enzyme can be improved by reducing the number of sulfhydryl groups. 14

15. 4. Single amino acid changes The stability and biological activity of recombinant proteins can be improved by a second generation variant . This is achieved by a single amino acid change. 5. Improving kinetic properties of enzymes The functional activities of enzyme can be improving their kinetic properties through oligonucleotide directed mutagenesis . This is required for enzymes having industrial and therapeutically benefits. 15

16. Application of protein engineering Protein engineering is used for cancer treatment studies . Pre-targeted radioimmune therapy is potential cancer treatment as pre targeting minimises radiation toxicity by spreading the rapidly cleared radionuclide and the long circulating antibody. protein engineering techniques are also used for producing therapeutic proteins . Therapeutic proteins are used to treat patients suffering from cancers, heart attacks, strokes, cystic fibrosis, diabetes, anemia and heamophilia. Protease, amylase and lipase are important enzymes for industrial food and detergent applications. Protease are commonly used for food industry in milk clotting, flavours and low allergic infant formulae. Lipase are used for stability and chees and flavours application in food industry 16

17. Amylases are commonly used for scarification of starch and bread softness in food industry. Protein engineering are used for improvement of microbial strains and their enzymes in bio-remediation applications. Redox protein and enzyme can be modified by protein engineering to be used as Nano devises for biosensing. The activity or properties of food-processing enzymes (amylase, lipase) are improved by protein engineering and rDNA technology. 17

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