Identification of phosphorylation sites of orphan response regulator CovR of Streptococcus suis serotype 2
WANG Qiao-qiao1,2, LI Chao-long1,2, ZHANG Hui-fang1,2, LIU Xu-miao1,2, ZHENG Feng2, WANG Chun-hui2, PAN Xiu-zhen1,2, CAO Xiang-rong1
1. College of Life Sciences, Nanjing Normal University, Nanjing 210023, China; 2. Hua Dong Research Institute for Medicine and Biotechnics, Nanjing 210002, China
Abstract: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 45th, 148th, 150th, 159th, 168th, 194th and 219th threonines; the 40th, 172th and 215th serines; and the 225th 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.
王悄悄, 李超龙, 张会芳, 刘旭苗, 郑峰, 汪春晖, 潘秀珍, 曹祥荣. 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[J]. 中国人兽共患病学报, 2021, 37(5): 392-397.
WANG Qiao-qiao, LI Chao-long, ZHANG Hui-fang, LIU Xu-miao, ZHENG Feng, WANG Chun-hui, PAN Xiu-zhen, CAO Xiang-rong. Identification of phosphorylation sites of orphan response regulator CovR of Streptococcus suis serotype 2. Chinese Journal of Zoonoses, 2021, 37(5): 392-397.
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