赤红球菌组氨酸激酶的融合表达和功能分析

对赤红球菌的组氨酸激酶基因进行密码子优化，将优化后的组氨酸激酶基因（rhks）构建重组表达质粒pGEX-4T-2-rhks。将此质粒导入到大肠杆菌BL21（DE3）中进行异源表达。在25 ℃和1 mmol/L异丙基-β-D-硫代吡喃半乳糖苷诱导条件下，组氨酸激酶融合蛋白（GST-RHK）获得成功表达，并具有催化活性。经谷胱甘肽琼脂糖亲和层析纯化，获得电泳纯的GST-RHK，其中纯化倍数为3.1，得率为19.5%。该蛋白大小约为72.75 kDa，Km、Vmax和Kcat值分别为20.92 μmol/L、0.17 μmol/（L·min）和1.4 min－1。野生型赤红球菌、组氨酸激酶基因增强株sdrhkE和组氨酸激酶基因敲减株sdrhkD在分别含有苯酚、甲苯、氯苯、异辛烷4 种有机溶剂的培养基中培养，菌株sdrhkD的生长情况都优于野生型赤红球菌，菌株sdrhkE的生长情况都低于野生型赤红球菌。本研究为进一步揭示赤红球菌SD3中组氨酸激酶涉及的信号转导途径与赤红球菌有机溶剂耐受性的关联机制提供一定参考依据。

Fusion Expression and Functional Analysis of Histidine Kinase from Rhodococcus ruber

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.