Zhao H, Giver L, Shao Z, Affholter J A, Arnold F H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena 91125, USA.
Nat Biotechnol. 1998 Mar;16(3):258-61. doi: 10.1038/nbt0398-258.
We have developed a simple and efficient method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) consists of priming the template sequence(s) followed by repeated cycles of denaturation and extremely abbreviated annealing/polymerase-catalyzed extension. In each cycle the growing fragments anneal to different templates based on sequence complementarity and extend further. This is repeated until full-length sequences form. Due to template switching, most of the polynucleotides contain sequence information from different parental sequences. The method is demonstrated by the recombination of two genes encoding thermostable subtilisins carrying two phenotypic markers separated by 113 base pairs and eight other point mutation markers. To demonstrate its utility for directed evolution, we have used StEP to recombine a set of five thermostabilized subtilisin E variants identified during a single round of error-prone PCR mutagenesis and screening. Screening the StEP-recombined library yielded an enzyme whose half-life at 65 degrees C is 50 times that of wild-type subtilisin E.
我们已经开发出一种用于多核苷酸序列体外诱变和重组的简单高效方法。交错延伸过程（StEP）包括对模板序列进行引物退火，随后进行变性和极短退火/聚合酶催化延伸的重复循环。在每个循环中，生长的片段基于序列互补性与不同的模板退火并进一步延伸。重复此过程直至形成全长序列。由于模板切换，大多数多核苷酸包含来自不同亲本序列的序列信息。通过重组两个编码耐热枯草杆菌蛋白酶的基因证明了该方法，这两个基因携带两个由113个碱基对分隔的表型标记以及其他八个点突变标记。为了证明其在定向进化中的效用，我们使用StEP重组了在一轮易错PCR诱变和筛选过程中鉴定出的一组五个热稳定枯草杆菌蛋白酶E变体。筛选StEP重组文库得到了一种酶，其在65℃下的半衰期是野生型枯草杆菌蛋白酶E的50倍。
