| Title | Construction of an inducible stable cell line for efficient incorporation of unnatural amino acids in mammalian cells |
| Authors | Zhang, Ziwei Xu, Huan Si, Longlong Chen, Yi Zhang, Bo Wang, Yan Wu, Yiming Zhou, Xueying Zhang, Lihe Zhou, Demin |
| Affiliation | Peking Univ, Sch Pharmaceut Sci, State Key Lab Nat & Biomimet Drugs, Beijing 100191, Peoples R China. |
| Keywords | Unnatural amino acid Mammalian cells Incorporation efficiency Engineered eRF1 STOP CODON RECOGNITION RELEASE FACTOR ERF1 GENETIC-CODE TRANSLATION TERMINATION ESCHERICHIA-COLI TRANSFER-RNA SUPPRESSION EXPRESSION PROTEINS RIBOSOME |
| Issue Date | 2017 |
| Publisher | BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS |
| Citation | BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS.2017,489(4),490-496. |
| Abstract | The genetic incorporation of unnatural amino acids (Uaas) with defined properties into proteins at designated sites represents an extremely powerful tool for protein engineering. However, the efficient incorporation of Uaas in response to the amber stop codon in mammalian cells remains a substantial challenge due to the competition from release factor 1(RF1). Addressing this challenge will greatly broaden the power and scope of this technology. Here, we chose the eRF1 mutant, which can selectively enhance Uaa incorporation in response to the amber codon without increasing the readthrough of the opal and ochre codons. Then, we developed an engineered stable cell line using a tetracycline-controlled inducible lentiviral system for the conditional expression of mutant eRF1, which can minimize the potential effect on normal translation termination. Using the eRF1-engineered cells, we provided a 2-fold improvement in the yield of protein containing a Uaa incorporated at a single site, with the protein yield approaching 90% of the wild-type control without the amber codon. Moreover, we achieved the successful incorporation of Uaas at four sites in various proteins at a measured level of 20%. (C) 2017 Elsevier Inc. All rights reserved. |
| URI | http://hdl.handle.net/20.500.11897/471583 |
| ISSN | 0006-291X |
| DOI | 10.1016/j.bbrc.2017.05.178 |
| Indexed | SCI(E) |
| Appears in Collections: | 药学院 天然药物与仿生药物国家重点实验室 |
