Introduction and Aim: Shiga Toxins are the major pathogenicity factors of enterohemorrhagic E. coli (EHEC). Stx are AB5 toxins, the A-subunit of which inhibits eukaryotic protein biosynthesis. The B-pentamer is responsible for binding to the receptor globotriaosylceramide. Since the A-subunit alone was already shown to be functional without its corresponding B-subunit, investigation and analysis of the mechanisms are of interest. Since mg amounts of toxin are needed for detailed analysis, current protocols of protein purification had to be optimized.
The aim of the current study was to increase the yield of the pure A-subunit to a final yield of 200-300 µg StxA2a per litre expression culture.
Materials and Methods: For the expression of StxA2a, the stxA2a gene was cloned into the vector pET45b(+) under the control of a T7 promotor, and transformed into E. coli BL21 (DE3) C43. In a first step the yield of StxA2a was optimized under different expression conditions (expression medium, expression time, expression temperature, IPTG concentrations). In a second step, StxA2a was purified with a single step purification using ion exchange chromatography.
Results: The optimal expression of StxA2a was achieved in terrific broth after an incubation of 24 h at 30°C. The expression of StxA2a was induced with 1 mM IPTG. Since the His-tag present on the StxA2a was not accessible for purification on a metal-ion affinity column, ion exchange chromatography was used. Following this one step purification protocol, a yield of 567 µg pure StxA2a per litre expression culture was achieved.
Summary: The optimization of the expression and purification of StxA2a leaded to a 5.5 times higher yield of pure StxA2a to former protocols. The purified StxA2a will be used for further studies on the cytotoxicity, the cellular uptake and the intracellular transport of StxA2a.