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Updated: Jun 15, 2026
Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System
Published on: August 1, 2016
Synthesis of a hetero subunit RNA modification enzyme by the wheat germ cell-free translation system
1Department of Materials Science and Biotechnology, Graduate School of Sciences and Engineering, Ehime University, Ehime, Japan.
Abstract:
Cell-free translation systems are a powerful tool for the production of many kinds of proteins. However, there are some barriers to improve the system in order to make it a more convenient approach. These include the fact that the production of proteins made up of hetero subunits is difficult. In this chapter, we describe the synthesis of yeast tRNA (m(7)G46) methyltransferase as a model protein. This enzyme catalyzes transfer of a methyl group from S-adenosyl-L-methionine to guanine at position 46 in tRNA and generates N(7)-methylguanine. Yeast tRNA (m(7)G46) methyltransferase is composed of two protein subunits, Trm8 and Trm82. To obtain the active Trm8-Trm82 complex, co-translation of both subunits is necessary. Preparation of mRNAs, in vitro synthesis and purification of the complex are explained in this chapter.
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