title: Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation.
abstract: 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 degrees 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.
authors.affiliation: Tsukuba Research Institute, Novartis Pharma KK, Ohkubo 8, Tsukuba 300-2611, Japan.
authors.forename: Norio
authors.initials: N
authors.lastname: Hamamatsu
authors.forename: Takuyo
authors.initials: T
authors.lastname: Aita
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Nomiya
authors.forename: Hidefumi
authors.initials: H
authors.lastname: Uchiyama
authors.forename: Motowo
authors.initials: M
authors.lastname: Nakajima
authors.forename: Yuzuru
authors.initials: Y
authors.lastname: Husimi
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Shibanaka
mesh_terms: D045247:Chryseobacterium / Q000201:enzymology* / Q000235:genetics; D019020:Directed Molecular Evolution; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors; D006358:Hot Temperature; D009154:Mutation; D000085822:Prolyl Oligopeptidases; D015202:Protein Engineering; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases