PEDS Advance Access originally published online on May 31, 2005
Protein Engineering Design and Selection 2005 18(6):265-271; doi:10.1093/protein/gzi028
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Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation
1Tsukuba Research Institute, Novartis Pharma KK, Ohkubo 8, Tsukuba 300-2611 and 2Department of Functional Materials Science, Saitama University, Saitama 338-8570, Japan 3Present address: Computational Biology Research Center, National Institute of Advanced Industrial Science and Technology, 2–43 Aomi, Koto-ku, Tokyo 135-0064, Japan
4 To whom correspondence should be addressed. E-mail: yasuhiko.shibanaka{at}pharma.novartis.com
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.
Keywords: additivity principles/directed evolution/prolyl endopeptidase/thermostability
Received October 22, 2004; revised March 30, 2005; accepted April 11, 2005.
Edited by Alan Fersht
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