1. Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.
- Author
-
Adachi J, Katsura K, Seki E, Takemoto C, Shirouzu M, Terada T, Mukai T, Sakamoto K, and Yokoyama S
- Subjects
- Codon, Terminator genetics, Codon, Terminator metabolism, Monoiodotyrosine genetics, Protein Biosynthesis, RNA, Transfer metabolism, Subcellular Fractions metabolism, Escherichia coli metabolism, Escherichia coli Proteins biosynthesis, Monoiodotyrosine metabolism, Peptide Termination Factors deficiency
- Abstract
Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.
- Published
- 2019
- Full Text
- View/download PDF