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

Gene. 1999 Sep 17;237(2):361-71. doi: 10.1016/s0378-1119(99)00331-5.

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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • Bacteriophage T7 / genetics*
  • Cloning, Molecular
  • Gene Expression
  • Genetic Vectors / genetics*
  • Histidine / genetics
  • Mycobacterium tuberculosis / genetics
  • Promoter Regions, Genetic
  • Pseudomonas aeruginosa / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / isolation & purification*
  • Recombinant Fusion Proteins / metabolism

Substances

  • Bacterial Proteins
  • LasI protein, Pseudomonas aeruginosa
  • Recombinant Fusion Proteins
  • Histidine