Expression and Functional Analysis of the Argonaute Protein of <i>Thermus thermophilus</i> (TtAgo) in <i>E. coli</i> BL21(DE3)
Jiani Xing,
Lixia Ma,
Xinzhen Cheng,
Jinrong Ma,
Ruyu Wang,
Kun Xu,
Joe S. Mymryk,
Zhiying Zhang
Affiliations
Jiani Xing
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Lixia Ma
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Xinzhen Cheng
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Jinrong Ma
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Ruyu Wang
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Kun Xu
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
Joe S. Mymryk
Department of Microbiology & Immunology, Oncology and Otolaryngology, The University of Western Ontario, London, ON N6A 3K7, Canada
Zhiying Zhang
Key Laboratory of Animal Genetics, Breeding and Reproduction of Shaanxi Province, College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
The prokaryotic Argonaute proteins (pAgos) have been reported to cleave or interfere with DNA targets in a guide-dependent or independent manner. It is often difficult to characterize pAgos in vivo due to the extreme environments favored by their hosts. In the present study, we expressed functional Thermus thermophilus pAgo (TtAgo) in E. coli BL21 (DE3) cells at 37 °C. Initial attempts to express TtAgo in BL21(DE3) cells at 37 °C failed. This was not because of TtAgo mediated general toxicity to the host cells, but instead because of TtAgo-induced loss of its expression plasmid. We employed this discovery to establish a screening system for isolating loss-of-function mutants of TtAgo. The E. colifabI gene was used to help select for full-length TtAgo loss of function mutants, as overexpression of fabI renders the cell to be resistant to the triclosan. We isolated and characterized eight mutations in TtAgo that abrogated function. The ability of TtAgo to induce loss of its expression vector in vivo at 37 °C is an unreported function that is mechanistically different from its reported in vitro activity. These results shed light on the mechanisms by which TtAgo functions as a defense against foreign DNA invasion.
