摘要
目的利用Escherichia 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pCDFDuet-1::covR及pCDFDuet-1::covR/stk,重组表达蛋白分别命名为CovR1与CovR2。通过蛋白磷酸化质谱技术检验CovR2蛋白的关键磷酸化位点。分别构建CovR苏氨酸突变体CovRT、丝氨酸突变体CovRS、酪氨酸突变体CovRY及丝/苏/酪氨酸全突变体CovRA,与STK共表达后分析其磷酸化状态,确定CovR磷酸化靶点。结果在E.coli BL21表达系统中成功表达并纯化CovR1与CovR2蛋白,Western blot分析发现CovR1无磷酸化信号,CovR2可以被磷酸化。进一步对CovR2磷酸化质谱检测发现其第45、148、150、159、168、194、219位苏氨酸,第40、172、215位丝氨酸以及第225位酪氨酸为关键磷酸化位点。对CovRT、CovRS、CovRY及CovRA突变体磷酸化状态检测,结果表明CovR2的丝/苏/酪氨酸均可发生磷酸化。结论在E.coli BL21中共表达STK与CovR可导致CovR丝/苏/酪氨酸磷酸化,通过质谱技术鉴定了CovR的磷酸化位点,提示CovR可能是STK的磷酸化调控靶点。
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.
作者
王悄悄
李超龙
张会芳
刘旭苗
郑峰
汪春晖
潘秀珍
曹祥荣
WANG Qiao-qiao;LI Chao-long;ZHANG Hui-fang;LIU Xu-miao;ZHENG Feng;WANG Chun-hui;PAN Xiu-zhen;CAO Xiang-rong(College of Life Sciences,Nanjing Normal University,Nanjing 210023,China;Hua Dong Research Institute for Medicine and Biotechnics,Nanjing 210002,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2021年第5期392-397,共6页
Chinese Journal of Zoonoses
基金
国家自然科学基金面上项目(No.82072256,No.81571965)
江苏省自然科学基金面上项目(No.BK20201129,No.BK20151091)联合资助。
