Monday, July 30, 2007 - 11:15 AM
S12

Protein Engineering by Structure-Guided Recombination

Yougen Li1, Christopher R. Otey2, D. Allan Drummond2, Andrew M Sawayama2, and Frances H Arnold2. (1) DuPont, Experimental Station E228/310A, Wilmington, DE 19808, (2) Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125

We used structure-guided recombination to create a synthetic protein family using three cytochrome P450s and seven crossovers chosen to minimize structural disruption. The library comprises at least 2,300 new, properly folded and catalytically active P450 heme proteins, which differ from any known sequence at an average of 72 and by as many as 109 amino acids. We then explored the relationship between sequence and stability by analyzing the thermostabilities of 204 and the sequences of 613 chimeric proteins from this family. We found that the thermostability of a chimeric protein is accurately predicted by assuming that its fragments make additive contributions to stability, and those contributions can be determined from the frequencies with which they appear in folded chimeras. Using this as the basis for new approaches to protein stabilization, we have generated 44 thermostable cytochrome P450 heme domains, whose half-lives of inactivation at 57 oC are up to 108 times that of the most stable parent. Differing from any known natural P450 by as many as 100 amino acids, the stable P450s are diverse, yet retain catalytic activity. Some are considerably more active than the parent enzymes on a spectrum of substrates. Among the novel functions they exhibit is the ability to produce authentic human drug metabolites. This stabilized and functional protein family provides a unique ensemble for industrial applications and for studying sequence-stability-function relationships.

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