Expression of the cloned gene for enterotoxin STb of Escherichia coli

Abstract

This study involved the construction of hybrid plasmids to produce heat-stable enterotoxin type II of Escherichia coli (STb). The translation of the open reading frame for the STb gene estA was demonstrated in several ways. Studies using in vivo labeling with [35S]cysteine demonstrated a radiolabeled protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the expected molecular weight of 5,000 for toxin STb. Insertion of translational or transcriptional termination signals into the Bglll site of the estA gene blocked the expression of estA. The estA gene was cloned into high-expression vector pKC30 downstream from the strong Pl promoter. Northern (RNA) blotting assays revealed a 10to 20-fold increase in mRNA produced by strain C600F(pKC30STb) over other STb-producing strains, compared with little or no increase in enterotoxin activity demonstrated by bioassay. The estA gene, with its own promoter and Shine-Delgarno region and a portion of the sequence for the signal peptide deleted, was also inserted under the control of the tac promoter. Even after induction of the tac promoter by addition of isopropyl-,I-D-thiogalactopyranoside, no biologic enterotoxin activity could be identified. Neutralizing antibodies to STb were produced in rabbits by using either a purified OmpF-STb-pi-galactosidase fusion protein or a 19-amino-acid synthetic STb peptide coupled to * Corresponding author. provided by C. H. Lee (15). All of the plasmids used are described in Table 1. The host strains were E. coli MH3000 and TK1046 for pORF1 derivatives (41). C600F, N99CI+, and N5151 were used as hosts for pKC30 and its derivatives (2, 31). E. coli JM103 was used as the host for pDR540 and its subclones (42). E. coli C strain 3456 was used as the host for pCHL6. P3, a wild-type strain of E. coli containing the gene for enterotoxin STb, estA, and 10405, a wild-type E. coli strain without estA, were used as controls in the bioassays. All of the strains used are listed in Table 2. Media. Bacteria were grown in tryptone yeast extract medium or minimal medium A supplemented with 0.25% glucose or 0.20% lactose. Clones were screened for lactose fermentation on MacConkey lactose plates or on 5-bromo-4-chloro-3-indolyl-4-D-galactopyranoside plates with or without isopropyl- ,-D-thiogalactopyranoside (IPTG). In vivo radiolabeling was performed in modified minimal medium A (0.004 M dipotassium phosphate, 0.0022 M potassium biphosphate, 0.0017 M trisodium citrate, 0.0076 M ammonium sulfate, 0.008 M magnesium sulfate, 0.01% yeast extract, 5% glucose, required growth factors). Ampicillin was used at 50 ,ug/ml, and tetracycline was used at 20 ,ug/ml. Materials. Enzymes were purchased from New England BioLabs, Inc., or Boehringer Mannheim Biochemicals and used as recommended by the suppliers. 5-Bromo-4-chloro-3-indolyl-4-D-galactopyranoside, IPTG, and p-aminophenyl-,B-D-thiogalactosidase were purchased from Sigma Chemical Co. o-Nitrophenyl-,B-D-galactopyranoside was supplied by Boehringer Mannheim Biochemicals. Oligonucleotides were sy...

Some properties of purified Escherichia coli heat-stable enterotoxin II

“…However, the antibody titer remained low despite several booster doses, and the serum could not neutralize STII activity. Lawrence et al (16) reported the production of anti-STII serum by the injection of a fusion protein (OmpF-STb fragment-1-galactosidase) or a 19-amino-acid peptide. The serum reacted weakly at low dilutions with the enterotoxin in the culture supernatant.…”

Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain

“…In vivo radiolabeling experiments with [35S]cysteine showed that culture supernatants contained a labeled band with a molecular weight of about 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which would represent STb (8,13). Similarly, Kupersztoch et al (11) identified the extracellular mature form of STb with a molecular weight of 5,200.…”

“…Other workers have produced antibodies against the fusion proteins protein A-STb (9) or OmpF-STb-p-galactosidase (13) or against the synthetic peptide coupled to keyhole limpet hemocyanin (13). However, such fusion proteins are poorly active or nonactive in pig intestinal loops (9,13). In addition, antibodies directed against such fusion proteins may demonstrate cross-reactivity with non-STh-producing strains.…”

Some infectious causes of diarrhea in young farm animals.

“…STb induces intestinal secretion in newborn pigs and weanling-age pigs (391) but has no apparent activity in mice. STb is produced by porcine)(K88-positive) ETEC (202), and the mechanism by which it stimulates the intestinal mucosa to secrete is unknown (187,391). STa is produced by K99, F41, and 987P ETEC strains.…”