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

Purification of proteins from Escherichia call under native conditions is o ften hampered by inclusion-body formation after overexpression from T7 prom oter-based expression vectors. This is probably due to the relatively high copy number of the ColEl-based expression vectors. To circumvent these prob lems, the low-copy-number pViet and pNam expression vectors were constructe d. These vectors contain the pSC101 origin of replication and allow the exp ression 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 d efective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were success fully tested by purification of three very insoluble proteins (RmlD, LasI a nd RhlI) under non-denaturing conditions, and all three proteins retained e nzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aer uginosa RhlI protein was subjected to more detailed analyses, which indicat ed that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionin e (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 RhlII; (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). (C) 1999 Els evier Science B.V. All rights reserved.