| Autor: |
Jiro Adachi, Kazushige Katsura, Eiko Seki, Chie Takemoto, Mikako Shirouzu, Takaho Terada, Takahito Mukai, Kensaku Sakamoto, Shigeyuki Yokoyama |
| Jazyk: |
angličtina |
| Rok vydání: |
2019 |
| Předmět: |
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| Zdroj: |
International Journal of Molecular Sciences, Vol 20, Iss 3, p 492 (2019) |
| Druh dokumentu: |
article |
| ISSN: |
1422-0067 |
| DOI: |
10.3390/ijms20030492 |
| Popis: |
Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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