Pikkemaat Mariël G, Janssen Dick B
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands.
Nucleic Acids Res. 2002 Apr 15;30(8):E35-5. doi: 10.1093/nar/30.8.e35.
Directed evolution techniques allow us to genuinely mimic molecular evolution in vitro. To enhance this imitation of natural evolutionary processes on a laboratory scale in even more detail, we developed an in vitro method for the generation of random deletions and repeats. The pairwise fusion of two fragments of the same gene that are truncated by exonuclease BAL-31 either at the 3' or 5' side results in a deletion or a repeat at the fusion point. Although in principle the method randomly covers the whole gene, it can also be limited to a predefined area in the sequence by controlling the level of the initial truncation. To test the procedure and to illustrate its potential, we used haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (DhlA) as a model enzyme, since the adaptation of this enzyme towards new substrates is known to occur via the generation of this type of mutation. The results show that the mutagenesis method presented here is an effective tool for accessing formerly unexplorable sequence space and can contribute to the success of future directed evolution experiments.
定向进化技术使我们能够在体外真正模拟分子进化。为了在实验室规模上更详细地增强对自然进化过程的这种模仿，我们开发了一种体外产生随机缺失和重复序列的方法。同一基因的两个片段在3'或5'端被核酸外切酶BAL-31截断后进行成对融合，会在融合点产生缺失或重复序列。虽然原则上该方法能随机覆盖整个基因，但通过控制初始截断水平，也可将其限制在序列中的预定义区域。为了测试该程序并说明其潜力，我们使用自养黄色杆菌GJ10的卤代烷脱卤酶（DhlA）作为模型酶，因为已知这种酶对新底物的适应性是通过产生此类突变发生的。结果表明，本文提出的诱变方法是进入以前无法探索的序列空间的有效工具，并且有助于未来定向进化实验的成功。
