Expression and purification of Bacillus thuringiensis Sigma K in Escherichia coli

Abstract:

[Objective] To express and purify the regulator protein Sigma K (σK) from Bacillus thuringiensis HD73 in Escherichia coli. [Methods] The ORF (open reading frame) of the sigK gene was amplified by PCR from Bt strain HD73, and then cloned into the vector pET21b to generate pETsigK. The pETsigK was transformed into BL21(DE3). The SDS-PAGE, nickel column affinity purification, Q-Sepharose fast flow column purification and electrophoretic mobility shift assay (EMSA) experiments were carried out to analyze the purity and binding activity of His-Sigma K with cry1Ac gene controlled by Sigma K in vitro. [Results] The 27 kD His-Sigma K was expressed. EMSA results showed that the His-Sigma K could bind to the promoter of cry1Ac gene, which is controlled by Sigma K. [Conclusion] The His-Sigma K protein was successfully expressed and purified. The purified His-Sigma K could bind to Sigma K-controlled promoter.

Key words:Bacillus thuringiensis, Sigma K protein, Biological activity, Transcriptional regulation