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Volume 139, Issue 3

Fusion of the Genes Encoding Heat-Stable Enterotoxin b (STb) and the Maltose-Binding Protein to Obtain Mature STb Enterotoxin Free

Abstract

The heat-stable enterotoxin b gene () of was fused to the gene for maltose-binding protein (). The gene was cloned into the pMAL-p vector using PCR. The construct consists of the signal sequence of maltose-binding protein, which directs the export of the fusion protein to the periplasm, and the maltose-binding protein fused to the STb polypeptide. A sequence encoding a factor Xa cleavage site is present between and . The fused genes are controlled by P, a strong inducible promoter. Following IPTG induction, the recombinant strain expressed a 47 kDa protein, which was easily purified from osmotic shock fluid by using preparative electrophoresis and electroelution. Cleavage of the fusion protein with factor Xa generated the maltose-binding protein (42 kDa) and a polypeptide of approximately 5 kDa, corresponding to the molecular mass of mature STb. A monospecific polyclonal rabbit antiserum raised against purified STb reacted in immunoblot with the fusion protein and the cleaved-off peptide. A positive response was observed when testing the osmotic shock fluid containing the fusion protein in a rat intestinal loop assay. On average, 3–4 mg of MBP-STb protein was recovered per litre of induced recombinant strain.

  • Received:
  • Accepted:
  • Revised:
  • Published Online:
© Society for General Microbiology 1993
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1993-03-01
2024-04-18
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Fusion of the Genes Encoding Escherichia Coli Heat-Stable Enterotoxin b (STb) and the Maltose-Binding Protein to Obtain Mature STb Enterotoxin
Microbiology 139, 631 (1993); https://doi.org/10.1099/00221287-139-3-631
/content/journal/micro/10.1099/00221287-139-3-631
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