Abstract:

【Objective】 Prokaryotic expression vector of STK1 from Setosphaeria turcica was constructed and expressed to obtain His-tagged protein. 【Method】 A pair of primers were designed according to multiple clone sites in prokaryotic expression vector pET28a(+) and STK1 which was obtained in authors’ previous experiment (GenBank accession: AY849317). The open reading frame of 1 071 bp was amplified by PCR from S. turcica and was linked with pET28a(+), and then transformed into the host E.coli strain DH5α.The fragment was conformed to the original sequence. It indicated that the expression vector pET28a(+)-STK1 was constructed successfully. The pET28a(+)-STK1 plasmid was taken and transformed into BL21 (DE3) for expression. Induced by IPTG at 37℃, the expression product of STK1 was identified by SDS-PAGE and Western blot. 【Result】 The expected protein Stk1 had been expressed successfully in the form of inclusion bodies. The molecular weight of protein was 40.8 kD, and the quantity produced reached the highest when it was induced with 1 mmol•L-1 IPTG for 9 h. Meanwhile, the expressed protein showed the antigenic characteristics of His-6 tagged protein. 【Conclusion】 STK1 in signal transduction pathway of mitogen activated protein kinase from S. turcica was successfully expressed in E.coli, thus laying a foundation for the preparation of STK1 polyclonal antibody and for the study of its function.

Key words: Setosphaeria turcica, STK1, prokaryotic expression, His-6 tagged protein