A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a single-promoter vector and native signal peptide

Biotechnol Appl Biochem. 2016 Jul;63(4):539-45. doi: 10.1002/bab.1398. Epub 2015 Jul 17.

Abstract

The entire stx1 region from Escherichia coli O157:H7, containing two open reading frames (stx1a and stx1b), was cloned into pET-32a with a single promoter. This region was transformed into E. coli TransB (DE3), which is a trxB and gor mutation strain. After expression in the E. coli periplasm in a completely soluble form, the rStx1 was purified and verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), ELISA, and Western blot analysis. Our rStx1 have Vero cell median cytotoxic dose (CD50 ) and median lethal dose (LD50 ) values of approximately 30 ng and 1.5 µg, respectively. The final yield of the purified rStx1 ranged from 2 to 3 mg/L by one-step nickel affinity gel column chromatography. This method is an easy approach to the large-scale preparation of Stx1 at a reasonable cost.

Keywords: native signal peptide; recombinant Shiga toxin 1(rStx1); single promoter.

MeSH terms

  • Animals
  • Chlorocebus aethiops
  • Female
  • Gene Expression
  • Genetic Engineering / methods*
  • Genetic Vectors / genetics*
  • Mice
  • Promoter Regions, Genetic / genetics*
  • Protein Sorting Signals*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / toxicity
  • Sequence Analysis
  • Shiga Toxin 1 / chemistry*
  • Shiga Toxin 1 / genetics*
  • Shiga Toxin 1 / isolation & purification
  • Shiga Toxin 1 / toxicity
  • Vero Cells

Substances

  • Protein Sorting Signals
  • Recombinant Proteins
  • Shiga Toxin 1