Smith Matthew A, Bedbrook Claire N, Wu Timothy, Arnold Frances H
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, United States.
ACS Synth Biol. 2013 Dec 20;2(12):690-6. doi: 10.1021/sb400010m. Epub 2013 Jun 3.
Noncontiguous recombination (NCR) is a method to identify pieces of structure that can be swapped among homologous proteins to create new, chimeric proteins. These "blocks" are encoded by elements of sequence that are not necessarily contiguous along the polypeptide chain. We used NCR to design a library in which blocks of structure from Hypocrea jecorina cellobiohydrolase I (Cel7A) and its two thermostable homologues from Talaromyces emersonii and Chaetomium thermophilum are shuffled to create 531,438 possible chimeric enzymes. We constructed a maximally informative subset of 35 chimeras to analyze this library and found that the blocks contribute additively to the stability of a chimera. Within two highly stabilizing blocks, we uncovered six single amino acid substitutions that each improve the stability of H. jecorina cellobiohydrolase I by 1-3 °C. The small number of measurements required to find these mutations demonstrates that noncontiguous recombination is an efficient strategy for identifying stabilizing mutations.
非连续重组（NCR）是一种识别可在同源蛋白之间交换以产生新的嵌合蛋白的结构片段的方法。这些“模块”由沿着多肽链不一定连续的序列元件编码。我们使用NCR设计了一个文库，其中来自嗜热毁丝霉的纤维二糖水解酶I（Cel7A）及其来自埃默森篮状菌和嗜热毛壳菌的两个耐热同源物的结构模块被改组，以产生531,438种可能的嵌合酶。我们构建了一个包含35个嵌合体的最大信息量子集来分析这个文库，发现这些模块对嵌合体的稳定性有累加贡献。在两个高度稳定的模块中，我们发现了六个单氨基酸取代，每个取代都使嗜热毁丝霉纤维二糖水解酶I的稳定性提高了1-3℃。找到这些突变所需的测量次数很少，这表明非连续重组是识别稳定突变的有效策略。
