Koga Yuichi, Yamane Tsuneo, Nakano Hideo
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan.
Methods Mol Biol. 2007;375:165-81. doi: 10.1007/978-1-59745-388-2_9.
The single-molecule PCR-linked in vitro expression (SIMPLEX) technology, which can directly link a single molecule of a gene to its encoding protein, has been used to engineer enantioselectivity of lipase from Burkhorderia cepacia KWI-56. A combinatorial mutation has been introduced only to four residues in the hydrophobic substrate-binding pocket of the enzyme based on a structural model of the substrate-enzyme complex. Such focused mutation library constructed by the SIMPLEX technology has been screened for an enantiomeric substrate. Some combinations of substitutions in the four positions of the lipase have been found as effective for changing the enantio-preference from the (S)-form of p-nitrophenyl-3-phenylbutyrate to the (R)-form. Here, we describe the detail procedure to construct such an exclusively in vitro protein library and a practical screening method based on enzymatic activity.
单分子PCR连接体外表达（SIMPLEX）技术能够直接将单个基因分子与其编码蛋白相连接，该技术已被用于改造洋葱伯克霍尔德菌KWI-56脂肪酶的对映体选择性。基于底物-酶复合物的结构模型，仅对该酶疏水底物结合口袋中的四个残基进行了组合突变。通过SIMPLEX技术构建的这种聚焦突变文库已针对对映体底物进行筛选。已发现脂肪酶四个位置上的某些取代组合可有效将对映体偏好从对硝基苯基-3-苯基丁酸酯的（S）型转变为（R）型。在此，我们描述构建此类纯体外蛋白质文库的详细步骤以及基于酶活性的实用筛选方法。
