Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.
Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.
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DOI：
10.1007/978-1-4939-7574-7_3
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发表时间：
2018
影响因子：
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通讯作者：
Eiko Seki;T. Yanagisawa;S. Yokoyama
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文献类型：
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作者：
Eiko Seki;T. Yanagisawa;S. Yokoyama
无细胞蛋白合成（Cell-free protein synthesis， CFPS）是一种将非规范氨基酸（non - canonical amino acids, ncAAs）结合到蛋白质中的有效方法。体外合成的性质使得在体内特定部位结合ncAAs时，可以使用有毒或减少细胞摄取的实验条件。使用高繁殖性RF-1缺失菌株B-60的大肠杆菌提取物（S30）。∆A: Z和B-95。∆A，利用methanosarcina mazei的正交tRNA和氨基酰基-tRNA合成酶（aaRS）对，我们建立了CFPS方法，用于高产高效的ncAAs的多重掺入。在本章中，我们描述了我们制备S30和正交tRNAPyland PylRS对的方法，以及两种用于ncAA掺入的CFPS协议。
Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using theEscherichia colicell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs fromMethanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyland PylRS pair, and two CFPS protocols for ncAA incorporation.