Improved synthesis of Salmonella typhimurium enterotoxin using gene fusion expression systems.
Salmonella enterotoxin (Stn) is a virulence factor in S. typhimurium strain Q1 that causes both fluid secretion in ligated intestinal loops of rabbits and elongation of Chinese hamster ovary (CHO) cells. High-level expression systems are needed to provide Stn in soluble form for detailed study of the biological activity of Stn. To maximize the synthesis and solubility of Stn, we systematically compared the production of native Stn synthesized with a T7 RNA polymerase/promoter system to that of two fusion proteins: glutathione S-transferase::Stn (Gst::Stn) and thioredoxin A::Stn (TrxA::Stn). The latter fusion protein expression systems resulted in a 64-fold increase in Gst::Stn and TrxA::Stn antigen concentration, as measured by specific anti-peptide antibodies in an enzyme-linked immunosorbent assay (ELISA). Most of the toxin derived using these vector systems was insoluble; however, the solubility of the TrxA::Stn antigen increased by at least 50-fold, with a concomitant increase in CHO cell elongation activity. In addition, stn gene expression was enhanced more than 50-fold by addition of 0.2-0.4 M NaCl to Luria-Bertani medium. The biological activity of Stn also was increased in the high-osmolarity medium. Consequently, the expression of stn may be regulated by DNA supercoiling.
Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston 77555-1019.
A K
AK
Chopra
A R
AR
Brasier
M
M
Das
X J
XJ
Xu
J W
JW
Peterson
D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D014764:Viral Proteins
D004768:Enterotoxins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; C082864:enterotoxin, Salmonella; D012965:Sodium Chloride; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases