关键词: 组氨酸激酶；赤红球菌；表达与纯化；有机溶剂耐受性

Abstract: In the present work, the histidine kinase gene from Rhodococcus ruber SD3 was codon-optimized, and the optimized histidine kinase gene (rhks) was applied to construct a recombinant expression plasmid pGEX-4T-2-rhks. Then, the plasmid was transformed into Escherichia coli BL21 (DE3) for heterologous expression. Under the induction conditions of 25 ℃ and 1 mmol/L IPTG, histidine kinase fusion protein (GST-RHK) was successfully expressed and found to have catalytic activity. GST-RHK was purified electrophoretical homogeneity by glutathione agarose affinity chromatography, with a purification fold of 3.1 and a yield of 19.5%. The protein’s molecular mass was estimated to be 72.75 kDa. Its Km, Vmax and Kcat were 20.92 μmol/L, 0.17 μmol/(L·min) and 1.4 min-1, respectively. The wild-type, rhk enhancement (sdrhkE) and rhk knockdown (sdrhkD) strains were cultured in a medium containing phenol, toluene, chlorobenzene and isooctane individually. sdrhkD grew better than did the wild-type strain, whereas the growth of sdrhkE was inferior than the wild-type strain. This study may pave the way for unveiling the relationship between the signal transduction pathway mediated by histidine kinase and organic solvent tolerance in R. ruber SD3.

Key words: histidine kinase; Rhodococcus ruber; expression and purification; organic solvent tolerance