2型猪链球菌二元信号系统孤儿调控因子CovR磷酸化位点鉴定

目的 利用E scherichia coli BL21原核表达系统共表达2型猪链球菌丝氨酸/苏氨酸激酶STK与孤儿调控因子CovR蛋白,通过质谱鉴定CovR蛋白磷酸化位点,探究STK对CovR的磷酸化作用.方法 构建CovR蛋白表达载体pCDF-Duet-1::covR及pCDFDuet-1::covR/stk,重组表...

Identification of phosphorylation sites of orphan response regulator CovR of Streptococcus suis serotype 2

The Escherichia coli BL21 prokaryotic expression system was used to co-express serine/threonine kinase STK and the orphan response regulator CovR of Streptococcus suis serotype 2. The phosphorylation sites of CovR were identified by mass spectrometry to investigate the relationship between STK and CovR phosphorylation. Plasmids pCDFDuet-1::covR and pCDFDuet-1::covR/stk, whose expression products were named CovR1 and CovR2, were constructed. Subsequently, the phosphorylation sites of CovR protein were determined with mass spectrometry. According to the results of mass spectrometry, the threonine mutant CovRT, serine mutant CovRS, tyrosine mutant CovRY and serine/threonine/tyrosine mutant CovRA were constructed separately. After co-expression with STK, the phosphorylation status of those mutants was analyzed to determine the phosphorylation targets. Our data revealed that CovR1 and CovR2 were successfully expressed and purified, and Western blot results showed that the CovR1 protein expressed alone was not phosphorylated, whereas the CovR2 protein co-expressed with STK was phosphorylated. Phosphorylation mass spectrometry detection showed that the 45^th, 148^th, 150^th, 159^th, 168^th, 194^th and 219^th threonines; the 40^th, 172^th and 215^th serines; and the 225^th tyrosine were essential phosphorylation sites of CovR2 protein. Detection of CovR mutant phosphorylation suggested that serines, threonines and a tyrosine of CovR2 can indeed be phosphorylated. In conclusion, co-expression of STK and CovR in E. coli BL21 resulted in phosphorylation of serine, threonine and tyrosine residues of CovR, and the phosphorylation sites of CovR were identified by mass spectrometry technology. Thus, CovR may be a phosphorylation regulation target of STK.