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Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
Directed evolution of multimeric proteins is enabled by dual-compensatory gene duplication
Rezwan Siddiquee1,2,3, Felicia Lie1,3, Taylor N Szyszka1,2,3
1School of Chemistry, The University of Sydney; Camperdown, NSW 2006, Australia.
Abstract:
Gene duplication has played a critical role in the evolutionary history of proteins, enabling complex multimers to emerge from simpler precursors. Yet in protein engineering, current methods for directed evolution do not exploit gene duplication, hampering access to the vast array of diverse variants that are only enriched in the presence of a wild-type copy. We establish a directed evolution strategy for multimeric proteins that harnesses gene duplication to compensate for metabolic burden and self-assembly fitness, allowing previously inaccessible variants to be enriched. Starting from a homomeric 240-mer capsid, gene duplication enables selection of both extreme homomeric variants and obligate heteromers. This strategy significantly expands engineering access to diverse high-performing variants, while also supporting a plausible model for evolutionary diversification of higher-order multimers in nature.
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