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. (17th July 2015)

• Record Type: Journal Article
• Authors: Tu, Wei; Li, Tao; Wang, Qin; Cai, Kun; Gao, Xiang; Wang, Hui
• Is Part Of: Biotechnology and applied biochemistry. Volume 63:Number 4(2016:Jul./Aug.)
• Page Start: 539
• Page End: 545
• Publication Date: 2015-07-17
• Subjects: recombinant Shiga toxin 1(rStx1) -- single promoter -- native signal peptide; Biotechnology -- Periodicals; Biochemical engineering -- Periodicals; Biochemistry -- Periodicals; Biochemistry -- Periodicals; Genetic Techniques -- Periodicals; Microbiological Techniques -- Periodicals; 660.6
• DOI: 10.1002/bab.1398
• Languages: English
• ISSNs: 0885-4513

Abstract

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.