Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation.
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.
Hamamatsu
Norio
N
Tsukuba Research Institute, Novartis Pharma KK, Ohkubo 8, Tsukuba 300-2611, Japan.
Aita
Takuyo
T
Nomiya
Yukiko
Y
Uchiyama
Hidefumi
H
Nakajima
Motowo
M
Husimi
Yuzuru
Y
Shibanaka
Yasuhiko
Y
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
D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases