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http://dx.doi.org/10.7845/kjm.2014.4012

Establishment of a Selection System for the Site-Specific Incorporation of Unnatural Amino Acids into Protein  

Edan, Dawood Salim (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Choi, Inkyung (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Park, Jungchan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Publication Information
Korean Journal of Microbiology / v.50, no.1, 2014 , pp. 1-7 More about this Journal
Abstract
Site-specific incorporation of unnatural amino acids (SSIUA) into protein can be achieved in vivo by coexpression of an orthogonal pair of suppressor tRNA and engineered aminoacyl-tRNA synthetase (ARS) that specifically ligates an unnatural amino acid to the suppressor tRNA. As a step to develop the SSIUA technique in Escherichia coli, here we established a new 2-step screening system that can be used for selecting an ARS variant(s) that ligates an unnatural amino acid to a suppressor tRNA. A positive selection system consists of chloramphenicol acetyl transferase gene containing an amber mutation at the $27^{th}$ residue, and efficiently concentrated amber suppressible ARS with a maximum enrichment factor of $9.0{\times}10^5$. On the other hand, a negative selection system was constructed by adding multiple amber codons in front of a lethal gene encoding the control of cell death B toxin (ccdB) which acts as an inhibitory protein of bacterial topoisomerase II. Amber suppression of ccdB by an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (TyrRS) and an amber suppressor tRNA significantly inhibits bacterial growth. This selection system was also able to efficiently remove amber suppressible ARS which could ligate natural amino acids to the suppressor tRNA. Thus, sequential combination of these two selection systems might be able to function as a powerful tool for selecting an ARS variant that specifically ligates an unnatural amino acid to the suppressor tRNA from an ARS mutant pool.
Keywords
control of cell death B (ccdB); selection system; unnatural amino acid incorporation;
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1 Santoro, S.W., Wang, L., Herberich, B., King, D.S., and Schultz, P.G. 2002. An efficient system for the evolution of aminoacyl-tRNA synthetase specificity. Nat. Biotechnol. 20, 1044-1048.   DOI   ScienceOn
2 Wang, L., Brock, A., Herberich, B., and Schultz, P.G. 2001. Expanding the genetic code of Escherichia coli. Science 292, 498-500.   DOI   ScienceOn
3 Wang, L., Brock, A., and Schultz, P.G. 2002. Adding L-3-(2-naphthyl) alanine to the genetic code of E. coli. J. Am. Chem. Soc. 124, 1836-1837.   DOI   ScienceOn
4 Wang, L., Zhang, Z., Brock, A., and Schultz, P.G. 2003. Addition of the keto functional group to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 100, 56-61.   DOI   ScienceOn
5 Pastrnak, M., Magliery, T.J., and Schultz, P.G. 2000. A new orthogonal suppressor tRNA/aminoacyl-tRNA synthetase pair for evolving an organism with an expanded genetic code. Helv. Chim. Acta 83, 2277-2286.   DOI
6 Chow, C.M. and RajBhandary, U.L. 1993. Saccharomyces cerevisiae cytoplasmic tyrosyl-tRNA synthetase gene. J. Biol. Chem. 268, 12855-12863.
7 Bernard, P. and Couturier, M. 1992. Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. J. Mol. Biol. 226, 735-745.   DOI
8 Chin, J.W., Martin, A.B., King, D.S., Wang, L., and Schultz, P.G. 2002. Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli. Proc. Natl. Acad. Sci. USA 99, 11020-11024.   DOI   ScienceOn
9 Furter R. 1998. Expansion of the genetic code: site-directed p-fluorophenylalanine incorporation in Escherichia coli. Protein Sci. 7, 419-426.
10 Kowal, A.K., Kohrer, C., and RahBhandary, U.L. 2001. Twenty-first aminoacyl-tRNA synthetase - suppressor tRNA pairs for possible use in site-specific incorporation of amino acid analogues into proteins in eukaryotes and in eubacteria. Proc. Natl. Acad. Sci. USA 98, 2268-2273.   DOI   ScienceOn
11 Lee, C.P. and RahBhandary, U.L. 1991. Mutants of Escherichia coli initiator tRNA that suppress amber codons in Saccharomyces cerevisiae and are aminoacylated with tyrosine by yeast extracts. Proc. Natl. Acad. Sci. USA 88, 11378-11382.   DOI   ScienceOn
12 Liu, D.R. and Schultz, P.G. 1999. Progress toward the evolution of an organism with an expanded genetic code. Proc. Natl. Acad. Sci. USA 96, 4780-4785.   DOI   ScienceOn
13 Loris, R., Dao-Thi, M.H., Bahassi, E.M., Van Melderen, L., Poortmans, F., Liddington, R., Couturier, M., and Wyns, L. 1999. Crystal structure of CcdB, a topoisomerase poison from E. coli. J. Mol. Biol. 285, 1667-1677.   DOI   ScienceOn
14 Bernard, P., Kezdy, K.E., Van Melderen, L., Steyaert, J., Wyns, L., Pato, M.L., Higgins, P.N., and Couturier, M. 1993. The F plasmid CcdB protein induces efficient ATP-dependent DNA cleavage by gyrase. J. Mol. Biol. 234, 534-541.   DOI   ScienceOn