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KMID : 0364820140500010001
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Korean Journal of Microbiology
2014 Volume.50 No. 1 p.1 ~ p.7
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Establishment of a Selection System for the Site-Specific Incorporation of Unnatural Amino Acids into Protein
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Dawood Salim Edan
Choi In-Kyung
Park Jung-Chan
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Abstract
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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 27th residue, and efficiently concentrated amber suppressible ARS with a maximum enrichment factor of 9.0 × 105. 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.
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KEYWORD
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control of cell death B (ccdB), selection system, unnatural amino acid incorporation
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