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 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).
Hoang
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TT
Department of Microbiology, Colorado State University, Fort Collins, CO, 80523-1677, USA.
Ma
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Stern
R J
RJ
McNeil
M R
MR
Schweizer
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HP
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
D001426:Bacterial Proteins; C081237:LasI protein, Pseudomonas aeruginosa; D011993:Recombinant Fusion Proteins; D006639:Histidine