Hamamatsu Norio, Aita Takuyo, Nomiya Yukiko, Uchiyama Hidefumi, Nakajima Motowo, Husimi Yuzuru, Shibanaka Yasuhiko
Tsukuba Research Institute, Novartis Pharma KK, Ohkubo 8, Tsukuba 300-2611, Japan.
Protein Eng Des Sel. 2005 Jun;18(6):265-71. doi: 10.1093/protein/gzi028. Epub 2005 May 31.
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.
我们已经开发出一种高效的优化技术——“偏向性突变组装”，用于改善诸如热稳定性等蛋白质特性。在该策略中，利用重叠延伸聚合酶链反应技术，使用来自野生型和表型有利突变基因的DNA片段构建突变体文库，其中野生型基因中组装的突变数量由突变DNA片段与野生型片段的混合比例随机控制。高混合比例会导致突变体组成偏向于多点突变体。作为一个案例研究，我们应用此策略来提高脑膜炎败血黄杆菌脯氨酰内肽酶的热稳定性，发现随着混合比例的增加，文库中热稳定突变体的比例也增加。如果热稳定突变体的比例增加，那么找到它们所需的筛选工作量应该会减少。实际上，在用高混合比例制备的文库中仅筛选了2000个突变体后，我们就分离出了一个突变体，其在60℃下的活性半衰期比野生型脯氨酰内肽酶长1200倍。我们的结果表明，有利突变的积极积累会导致突变体文库质量的提高以及找到优良突变体所需筛选工作量的减少。
