Semisynthetic tRNA complement mediates in vitro protein synthesis

Cui, Zhenling, Stein, Viktor, Tnimov, Zakir, Mureev, Sergey and Alexandrov, Kirill (2015) Semisynthetic tRNA complement mediates in vitro protein synthesis. Journal of the American Chemical Society, 137 13: 4404-4413. doi:10.1021/ja5131963


 
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Author Cui, Zhenling
Stein, Viktor
Tnimov, Zakir
Mureev, Sergey
Alexandrov, Kirill
Title Semisynthetic tRNA complement mediates in vitro protein synthesis
Formatted title
Semisynthetic tRNA complement mediates in vitro protein synthesis
Journal name Journal of the American Chemical Society   Check publisher's open access policy
ISSN 1520-5126
0002-7863
Publication date 2015-04-08
Year available 2015
Sub-type Article (original research)
DOI 10.1021/ja5131963
Open Access Status Not Open Access
Volume 137
Issue 13
Start page 4404
End page 4413
Total pages 10
Place of publication Washington, DC United States
Publisher American Chemical Society
Language eng
Formatted abstract
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

 
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