嗜水气单胞菌十一碳焦磷酸合成酶XreF的原核表达及分离纯化

Prokaryotic Expression and Purification of Undecaprenyl Pyrophosphate Synthetase from Aeromonas hydrophila

目的:构建嗜水气单胞菌十一碳焦磷酸合成酶XreF基因的原核表达载体,转入大肠杆菌诱导表达高活性的XreF蛋白。方法:从嗜水气单胞菌中提取基因组DNA,以基因组DNA为模板进行PCR扩增,构建pPROEX-HTaXreF重组载体,转入大肠杆菌BL21(DE3)表达目的蛋白;用镍离子亲和层析、离子交换层析、凝胶过滤层析分离纯化目的蛋白XreF;用分析型凝胶过滤柱检测XreF的聚集状态。结果:构建了重组载体pPROEX-HTa-XreF,测序结果与XerF基因编码序列一致;XreF蛋白在大肠杆菌中经IPTG诱导高效表达,纯化获得高浓度(17.5 mg/mL)、高纯度(96%以上)的XreF蛋白;分析型凝胶过滤结果显示,XreF在溶液中为单体,在镁离子存在的情况下为二聚体。结论:获得并纯化了在大肠杆菌中高效表达的嗜水气单胞菌XreF,镁离子可诱导XreF在溶液中由单体向二聚体转化。

Objective:To construct a prokaryotic expression vector carrying undecaprenyl pyrophosphate synthetase XreF gene and transfer it into E.coli to induce XreF with high activity.Methods:The gene encoding XreF was amplified from genomic DNA extracted from Aeromonas hydrophila.The recombinant plasmid pPROEX-HTa-XreF was constructed and transferred into E.coli BL21(DE3)for expression of the target protein.Then XreF protein was separated and purified by nickel ion affinity chromatography,ion exchange chromatography and gel filtration chromatography.The polymeric state of XreF was detected using an analytical gel filtration column.Results:The XreF pPROEX-HTa-XreF recombinant vector was constructed,confirmed by gene sequence encoding XerF.The overexpression of XreF protein was induced by IPTG.XreF protein was purified and concentrated to17.5mg/mL of high purity(over96%).Analytical gel filtration showed that XreF prefers monomeric state in solution and dimeric state in the presence of magnesium ions.Conclusion:The overexpressed A.hydrophila XreF in E.coli was obtained and purified.Magnesium ions can induce the transform of XreF from monomeric state to dimer state in solution.