Cell-free synthesis system suitable for disulfide-containing proteins
Cell-free synthesis system suitable for disulfide-containing proteins
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DOI：
10.1016/j.bbrc.2012.12.107
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发表时间：
2013-02-08
影响因子：
3.1
通讯作者：
Kigawa, Takanori
中科院分区：
文献类型：
--
作者：
Matsuda, Takayoshi;Watanabe, Satoru;Kigawa, Takanori
许多重要的治疗靶点是具有多个二硫键的分泌蛋白，如抗体、细胞因子、激素和蛋白酶。利用基于细胞的表达系统制备用于结构和功能分析的这些蛋白质仍然存在一些问题，如效率低、产率低以及难以进行稳定同位素标记。无细胞(或体外)蛋白质合成系统已成为一种有用的蛋白质生产方法。无细胞体系的开放性允许直接控制反应环境以促进蛋白质折叠，使其非常适合合成含二硫键的蛋白质。在这项研究中，我们开发了基于大肠杆菌细胞裂解物的含二硫键蛋白质的无细胞合成系统，该系统可以产生足够数量的功能蛋白质用于核磁共振分析。谷胱甘肽缓冲液的使用促进了二硫键的形成。此外，二硫键异构酶DsbC在蛋白质合成反应中催化了错误形成的二硫键的有效洗牌。我们成功地合成了毫克量N-15标记的高级真核蛋白--牛胰蛋白酶抑制因子(BPTI)和人溶菌酶C(LYZ)。核磁共振波谱和功能分析表明，合成的蛋白质既具有催化功能，又能正确折叠。因此，无细胞体系对于合成用于结构和功能分析的含二硫键蛋白质是有用的。(C)2013 Elsevier Inc.保留所有权利。
Many important therapeutic targets are secreted proteins with multiple disulfide bonds, such as antibodies, cytokines, hormones, and proteases. The preparation of these proteins for structural and functional analyses using cell-based expression systems still suffers from several issues, such as inefficiency, low yield, and difficulty in stable-isotope labeling. The cell-free (or in vitro) protein synthesis system has become a useful protein production method. The openness of the cell-free system allows direct control of the reaction environment to promote protein folding, making it well suited for the synthesis of disulfide-containing proteins. In this study, we developed the Escherichia coli (E. coli) cell lysate-based cell-free synthesis system for disulfide-containing proteins, which can produce sufficient amounts of functional proteins for NMR analyses. Disulfide bond formation was facilitated by the use of glutathione buffer. In addition, disulfide isomerase, DsbC, catalyzed the efficient shuffling of incorrectly formed disulfide bonds during the protein synthesis reaction. We successfully synthesized milligram quantities of functional N-15-labeled higher eukaryotic proteins, bovine pancreatic trypsin inhibitor (BPTI) and human lysozyme C (LYZ). The NMR spectra and functional analyses indicated that the synthesized proteins are both catalytically functional and properly folded. Thus, the cell-free system is useful for the synthesis of disulfide-containing proteins for structural and functional analyses. (C) 2013 Elsevier Inc. All rights reserved.