미생물학회지 (Korean Journal of Microbiology)

  • 제50권1호
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  • Pages.1-7
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  • 2014
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  • 0440-2413(pISSN)
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  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
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비천연 아미노산의 위치특이적 단백질 삽입을 위한 Amino Acyl-tRNA Synthetase 선별시스템 개발

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)
  • 투고 : 2014.03.11
  • 심사 : 2014.03.19
  • 발행 : 2014.03.31
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초록

생명체에서 비천연 아미노산을 단백질의 특정 위치에 삽입하는 방법으로 orthogonal suppressor tRNA와 여기에 비천연 아미노산을 특이적으로 결합시킬 수 있는 유전자 변형된 aminoacyl-tRNA synthetase (ARS)가 활용되고 있다. 이 기술개발을 위해서는 돌연변이를 유발한 ARS library로부터 비천연 아미노산만을 특이적으로 결합시킬 수 있는 변형된 ARS를 탐색하기 위한 선별시스템이 필요하다. 본 논문에서는 대장균에서 작용하는 2단계로 구성된 새로운 선별시스템을 개발하였다. 먼저 양성선별 시스템은 27번 잔기를 amber 코돈으로 치환한 Chloramphenicol acetyl transferase 유전자로 구성되어 있으며, 이유전자의 amber suppression에 의해 chloramphenicol 배지에서 생존함에 따라 활성을 나타내는 ARS를 최고 $9.0{\times}10^5$배로 농축할 수 있었다. 반면 음성선별 시스템은 대장균의 Topoisomerase II의 기능을 억제하는 단백질을 암호화하는 control of cell death B (ccdB) 유전자의 N-말단 앞에 3개의 amber 코돈을 삽입하여 제작하였다. 이 음성선별 시스템을 가진 대장균에 orthogonal pair인 Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Scc TyrRS)와 amber suppressor tRNA를 형질전환하면 amber suppression으로 CcdB가 발현되어 대장균의 성장이 억제되는 것을 확인하였으며, 천연 아미노산에 대한 특이성을 가진 ARS를 효과적으로 제거하는 것을 관찰하였다. 따라서, 양성선별 및 음성선별 시스템을 순차적으로 거침으로써 무작위적으로 아미노산에 대한 특이성을 변형시킨 ARS 라이브러리로부터 비천연 아미노산을 suppressor tRNA에 특이적으로 결합하는 유전자 변형 ARS를 탐색하는데 유용하게 사용될 수 있을 것이다.

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.

키워드

참고문헌

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피인용 문헌

  1. Improving amber suppression activity of an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase and a variant of E. coli initiator tRNA, fMam tRNACUA, for the efficient incor vol.54, pp.4, 2014, https://doi.org/10.7845/kjm.2018.8101