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Gene. 1999 Sep 17;237(2):361-71.

Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of mycobacterium tuberculosis RmlD and pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI.

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  • 1Department of Microbiology, Colorado State University, Fort Collins, CO, 80523-1677, USA.

Abstract

Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).

PMID:
10521660
[PubMed - indexed for MEDLINE]
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