title: 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.
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).
authors.affiliation: Department of Microbiology, Colorado State University, Fort Collins, CO, 80523-1677, USA.
authors.forename: T T
authors.initials: TT
authors.lastname: Hoang
authors.forename: Y
authors.initials: Y
authors.lastname: Ma
authors.forename: R J
authors.initials: RJ
authors.lastname: Stern
authors.forename: M R
authors.initials: MR
authors.lastname: McNeil
authors.forename: H P
authors.initials: HP
authors.lastname: Schweizer
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics*; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000235:genetics; D011401:Promoter Regions, Genetic; D011550:Pseudomonas aeruginosa / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C081237:LasI protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D006639:Histidine