1. Protein Engineering BIT 230

2. Protein Engineering Design and construction of proteins by recombinant DNA techniques Michael Smith developed using oligonucleotide (site- directed) mutagenesis

3. Mutagenesis:Why Mutate? Native proteins are not well suited for industrial application Native proteins are not optimized for medicinal purposes Increase the efficiency of enzyme-catalyzed reactions Eliminate the need for cofactor in enzymatic reaction Change substrate binding site to increase specificity Change the thermal tolerance Change the pH stability Increase proteins resistance to proteases (purification) Signal sequences - secretion rare codon changes

4. Aspargine Changes If asparagine and glutamine present in protein when heated, ammonia is released amino acids convert to aspartic acid and glutamic acid Protein may refold LOSE ACTIVITY

5. Adding Disulfide Bonds Usually found in extracellular proteins, not intracellular Cross link between chains or in chains formed by oxidation of cysteine residues connective tissue fibrin blood clots Artificial addition may increase stability of protein Avoid active site (enzyme) Example: xylanase used to treat wood pulp in paper production needs to function at high temp

6. Reducing Free Sulfhydryl Residues Cysteine residues may cause dimerization through intermolecular disulfide bonding Convert Cys to another amino acid (serine?) reduce dimerization maintain activity of enzyme

7. Enzyme Activity and Specificity Increase enzymatic activity by increasing affinity for enzyme change sequences in substrate binding site Change substrate of enzyme tPA tissue plasminogen activator dissolves blood clots

8. Test, Test, Test