期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 55, 期 2, 页码 325-333出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.06.014
关键词
T7 expression; protein expression; T7 RNA polymerase; T7 lysozyme
类别
资金
- NCRR NIH HHS [P20RR018727, P20 RR018727] Funding Source: Medline
- NIAID NIH HHS [R03 AI065852, R03 AI065852-01A1] Funding Source: Medline
The T7-expression system has been very useful for protein expression in Escherichia coli. However, it is often desirable to over-express proteins in species other than E coli. Here, we constructed an inducible broad-host-range T7-expression transposon, which allows simple one-step construction of T7-expression strains in various species, providing the option to over-express proteins of interest in a broader host-range. This transposon contains the T7 RNA polymerase driven by the lacUV5 promoter, which is repressed by the lac-repressor. Leaky expression is prevented by the presence of T7-lysozyme on this construct. The complete T7-expression system is flanked by mariner transposon repeats of the suicidal R6K gamma ori plasmid, pBT20-Delta bla. Stable integration of the whole system is possible by a one-step selection for a Flp-excisable Gm R-marker. We showed the engineering of E coli, Pseudomonas aeruginosa, Erwinia carotovora, Salmonella choleraesuis, Agrobacterium tumefaciens, and Chromobacterium violaceum strains with this construct and demonstrated the expression of the Burkholderia pseudomallei Asd protein in these hosts, by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG). (C) 2007 Elsevier Inc. All rights reserved.
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