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Updated: Apr 11, 2026
Overexpressing and Purifying a Toxic Nuclease from Escherichia coli
Published on: August 29, 2025
A simple method for expression and purification of Shiga toxin 1 (Stx1) with biological activities by using a
1State Key Laboratory of Pathogens and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Fengtai District, Beijing, People's Republic of China.
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.
