Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes
Implementing bacterial acid resistance into cell-free protein synthesis for buffer-free expression and screening of enzymes
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DOI：
10.1002/bit.25671
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发表时间：
2015-12-01
影响因子：
3.8
通讯作者：
Kim, Dong-Myung
中科院分区：
文献类型：
--
作者：
Kim, Ho-Cheol;Kim, Kwang-Soo;Kim, Dong-Myung
无细胞蛋白质合成利用从细胞中分离出来的翻译机制来体外表达模板基因。由于它不需要基因克隆和细胞培养步骤就能产生蛋白质，因此无细胞蛋白质合成可以作为酶文库高通量表达的通用平台。此外，无细胞蛋白质合成的开放性允许酶合成与后续筛选步骤直接整合。然而，传统的反应混合物中存在高浓度的化学缓冲液，这使得基于pH的合成酶的分析很难简化无细胞蛋白质的合成。在这项研究中，我们将一种酶辅助细菌耐酸机制应用到基于大肠杆菌提取物的无细胞蛋白质合成系统中，取代了化学缓冲液。工程谷氨酸脱羧酶(GAD)通过消耗质子将谷氨酸转化为氨基丁酸(GABA)，应用于无细胞合成酶的反应混合物中，能够在酶合成过程中保持反应混合物的pH。由于谷氨酸耗尽后反应混合物失去缓冲能力，合成的酶无需纯化即可直接检测。所设计的方法成功地应用于唾液酸基转移酶基因突变文库的筛选，以筛选出酶活性提高的突变株。生物技术。比昂斯。2015；112：2630-2635。(C)2015年威利期刊公司。
Cell-free protein synthesis utilizes translational machinery isolated from the cells for in vitro expression of template genes. Because it produces proteins without gene cloning and cell cultivation steps, cell-free protein synthesis can be used as a versatile platform for high-throughput expression of enzyme libraries. Furthermore, the open nature of cell-free protein synthesis allows direct integration of enzyme synthesis with subsequent screening steps. However, the presence of high concentration of chemical buffers in the conventional reaction mixture makes it difficult to streamline cell-free protein synthesis with pH-based assay of the synthesized enzymes. In this study, we have implemented an enzyme-assisted bacterial acid resistance mechanism into an Escherichia coli (E.coli) extract-based cell-free protein synthesis system in place of chemical buffers. When deployed in the reaction mixture for cell-free synthesis of enzymes, through proton-consuming conversion of glutamate into -aminobutyric acid (GABA), an engineered glutamate decarboxylase (GAD) was able to maintain the pH of reaction mixture during enzyme synthesis. Because the reaction mixture becomes free of buffering capacity upon the depletion of glutamate, synthesized enzyme could be directly assayed without purification steps. The designed method was successfully applied to the screening of mutant library of sialyltransferase genes to identify mutants with improved enzymatic activity. Biotechnol. Bioeng. 2015;112: 2630-2635. (c) 2015 Wiley Periodicals, Inc.