牛种布鲁氏菌ArsR2蛋白的原核表达及磷酸化位点鉴定

Prokaryotic Expression and Phosphorylation Site Identification of ArsR2 Protein of Brucella abortus

用原核表达系统表达并纯化牛种布鲁氏菌S2308株ArsR2蛋白,软件分析该蛋白相关信息,质谱鉴定该蛋白修饰位点。以牛种布鲁氏菌S2308株基因组为模板,通过PCR扩增arsR2基因,构建pET-32a-ArsR2重组质粒,经测序正确后,转化至大肠埃希氏菌感受态细胞BL21(DE3)中进行IPTG诱导表达,通过Ni-NTA亲和层析法进行ArsR2蛋白的纯化,并进行SDS-PAGE和Western blot鉴定;对牛种布鲁氏菌ArsR2蛋白的理化性质和磷酸化位点进行预测,通过质谱鉴定ArsR2蛋白的修饰位点。结果显示,成功表达并纯化ArsR2蛋白,ArsR2蛋白无跨膜区,存在磷酸化修饰,质谱鉴定出ArsR2蛋白具有2个磷酸化位点。表明原核表达的牛种布鲁氏菌S2308株ArsR2蛋白存在磷酸化修饰位点,为探究该蛋白的生物学功能奠定了基础。

The purpose of this study was to express and purify the ArsR2 protein of Brucella abortus S2308 strain by prokaryotic expression system,analyze the related information of the protein by bioinformatics software,and identify the modification site of the protein by mass spectrometry.The arsR2 gene was amplified by PCR using the genome of Brucella abortus S2308 strain as a template,and the pET-32a-ArsR2 recombinant plasmid was constructed.After sequencing,the recombinant plasmid was transformed into E.coli competent cells BL21(DE3)for expression under induction of IPTG.The ArsR2 protein was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blot.The physical and chemical properties and phosphorylation sites of ArsR2 protein of Brucella abortus were predicted by bioinformatics methods.On this basis,the modification sites of ArsR2 protein were identified by mass spectrometry.The results showed that ArsR2 protein was successfully expressed and purified.Through bioinformatics analysis,it was found that ArsR2 protein had no transmembrane region,and phosphorylation modification.The ArsR2 protein was further identified by mass spectrometry to have two phosphorylation sites.The results showed that the ArsR2 protein of Brucella abortus S2308 strain expressed in prokaryotic expression had phosphorylation modification sites,which laid a foundation for further exploring the biological function of the protein.