Mol Biotech Ch8

Mol Biotech Ch8 - Chapter 8: Protein Engineering Molecular...

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Chapter 8: Protein Engineering Molecular Biotechnology Most mutations reduce enzyme activity. You need to target a specific trait and screen through all the losers to find a winner for just that trait. E.g., substrate binding (Km), catalytic rate (Vmax), thermal or pH tolerance, activity in organic solvents, eliminating cofactors, substrate specificity, or protease resistance. There are two types of strategies in mutagenesis: Directed vs. random. With directed mutagenesis, you have some knowledge of the 3D structure of the protein and make a limited number of specific changes. With random mutagenesis, you have no knowledge of the protein’s structure, so you resign yourself to creating a huge number of mutations quickly over the whole target region and then screening through all those mutants. Fig. 8.1: Introduce a single base change with a primer carrying that base change. Clone your target DNA into the ssDNA phage, M13. Bind your primer to ssDNA isolated from the virus particle (virion). Polymerize the 2 nd strand in a test tube ( in vitro ) to create dsDNA (M13 virus has both ds and ssDNA stages to its life cycle). As the virus multiplies in E. coli in its dsDNA phase, should have half of the dsDNA circles contain the mutation and half not. These replicate to form ssDNA circles that are secreted in virions. In actual fact, only 1-5% of the circles contain the mutation. Fig. 8.2: To improve the % yield of mutant dsDNA, grow out template DNA in an E. coli with the dut mutation, which increases uracil incorporation, and with the ung mutation, which deactivates uracil removal from DNA. The result is an M13 template with uracil in it. Go though the same protocol as in Fig. 8.1, in an E. coli with the wild type versions of dut / ung . Most parental strands are then degraded by E. coli at the sites of uracil incorporation, while the mutated strand serves as the template for M13 replication. Fig. 8.3: You can generate ssDNA template by just treating normal plasmids with alkali. Then add three mutagenic primers. One is a custom primer that puts your mutation into the target gene. The other two change the antibiotic resistance (AbR) genes from amplicillin sensitive (ampS) to ampicillin resistant (ampR) and tetR to tetS. This makes it easy to screen for the mutated strand (ampR/tetS), avoiding the parental strand
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Mol Biotech Ch8 - Chapter 8: Protein Engineering Molecular...

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