Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains

Methods Mol Biol. 2018:1728:49-65. doi: 10.1007/978-1-4939-7574-7_3.

Abstract

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 the Escherichia coli cell 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 from Methanosarcina 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 tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Keywords: Aminoacyl-tRNA synthetase; Cell extract; Cell-free protein synthesis; Noncanonical amino acids; Release factor 1 (RF-1); S30; tRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acyl-tRNA Synthetases / metabolism
  • Cell Extracts
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism*
  • Gene Deletion*
  • Peptide Termination Factors / genetics*
  • Protein Biosynthesis*
  • RNA, Transfer / metabolism

Substances

  • Cell Extracts
  • Peptide Termination Factors
  • peptide-chain-release factor 3
  • RNA, Transfer
  • Amino Acyl-tRNA Synthetases