| Autor: |
Hamamatsu, Norio, Aita, Takuyo, Nomiya, Yukiko, Uchiyama, Hidefumi, Nakajima, Motowo, Husimi, Yuzuru, Shibanaka, Yasuhiko |
| Zdroj: |
Protein Engineering Design and Selection; June 2005, Vol. 18 Issue: 6 p265-271, 7p |
| Abstrakt: |
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. |
| Databáze: |
Supplemental Index |
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