Biotechnology and Bioprocess Engineering 2023; 28(3): 398-405  
Development of Orthogonal Aminoacyl tRNA Synthetase Mutant with Enhanced Incorporation Ability with Para-azido-L-phenylalanine
Dongheon Lee, Min-Kyu Kim, and Jong-il Choi
Dongheon Lee, Jong-il Choi*
Department of Biotechnology and Bioengineering, Chonnam National University, Gwangju 61186, Korea
Tel: +82-62-530-1846; Fax: +82-62-530-1949
Min-Kyu Kim
Radiation Research Division, Korea Atomic Energy Research Institute, Jeongeup 56212, Korea
Received: August 25, 2022; Revised: November 11, 2022; Accepted: November 17, 2022; Published online: June 30, 2023.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Orthogonal aminoacyl tRNA synthetase (aaRS)/ tRNA pairs are an efficient tool for the site-specific introduction of para-azido-L-phenylalanine (pAzF), a noncanonical amino acid, into the amber codon of proteins. In the present study, to improve amber suppression by aaRS, random mutagenesis using error-prone polymerase chain reaction method was carried out, and mutants with enhanced pAzF-incorporation ability were selected using fluorescence-activated cell sorting (FACS). For screening, the mutant superfolder green fluorescent protein with an amber codon was used as the reporter. Furthermore, the predicted structure of resulting aaRS mutants were analyzed. Three aaRS mutants — K776, K801, and M320 — showed 2.9-, 1.5-, and 3.7-times greater pAzF-incorporation ability, respectively, than did the control. The mutants also exhibited increased selectivity for pAzF introduction. The predicted protein models for the aaRS mutants showed that mutations in the anticodon and acceptor stem of the tRNA recognition region affected the tRNA binding affinity. These results demonstrate that random mutagenesis can cover the missed possibility of rational design or site directed mutagenesis, and screening with FACS is an efficient and rapid method for the evolution of aaRS. It can be also utilized for the development of other proteins with a variety of non-canonical amino acids.
Keywords: aminoacyl tRNA synthetase, non-canonical amino acid, evolution, protein modeling

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