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MIT OpenCourseWare http://ocw.mit.edu 7.344 Directed Evolution: Engineering Biocatalysts Spring 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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Session 11 Lecture Notes 1. The authors’ primarily address transformation efficiencies as limiting steps. Cytotoxocity is mentioned as well as the expression hosts’ ability to escape selection pressure by increased protein expression or alternative metabolism. If your downstream application is in vivo these points are useless. 2. Ribosome display is the strategy. See Figure 1. The authors use an enrichment for activity based on the binding of active site inhibitors – see figure 2 for a mechanistic explanation. A linker protein is fused to the enzyme to allow for proper protein folding and reactivity. Biotin is tethered to the inhibitor allowing for pulldown of active complexes. Dissociation of the complex with RNAse and sequencing (RT PCR) is used to identify useful mutants.
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