PEDS Advance Access published online on May 31, 2005
Protein Engineering Design and Selection, doi:10.1093/protein/gzi028
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1 Tsukuba Research Institute, Novartis Pharma KK, Ohkubo 8, Tsukuba 300-2611, Japan
* To whom correspondence should be addressed. We have developed an efficient optimization technique, ‘biased mutation-assembling’, for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60°C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.
Received October 22, 2004
Revised March 30, 2005
Accepted April 11, 2005
Article
Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation
2 Department of Functional Materials Science, Saitama University, Saitama 338-8570, Japan
Yasuhiko Shibanaka, E-mail: yasuhiko.shibanaka{at}pharma.novartis.com
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