Das T, Schuster A, Schneider-Schaulies S, Banerjee A K
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195, USA.
Virology. 1995 Aug 1;211(1):218-26. doi: 10.1006/viro.1995.1394.
The phosphoprotein P gene of measles virus (Edmonston strain) has been cloned in the Escherichia coli expression vector pET-3a with a histidine tag at the C-terminal end. The expressed protein was soluble, unphosphorylated, and constituted 10 to 20% of total cellular protein. Recombinant P protein purified by Ni-affinity chromatography was found to be efficiently phosphorylated in vitro by recombinant casein kinase II (CKII) or by the CKII activity present in the uninfected cell extract. A comparison of phosphopeptide analyses between the in vivo- and the in vitro-32P-labeled P proteins revealed that both proteins share common phosphorylation sites. In an attempt to identify the exact site of the CKII-mediated phosphorylation, we altered specific serine residues located within the CKII consensus motif to alanine by site-directed mutagenesis. The results indicate that Ser 86, Ser 151, and Ser 180 located within the N-terminal half of the P protein are involved in the CKII-mediated phosphorylation of the P protein.
麻疹病毒（埃德蒙斯顿株）的磷蛋白P基因已被克隆到大肠杆菌表达载体pET-3a中，其C末端带有组氨酸标签。表达的蛋白是可溶的、未磷酸化的，占细胞总蛋白的10%至20%。通过镍亲和层析纯化的重组P蛋白在体外被重组酪蛋白激酶II（CKII）或未感染细胞提取物中存在的CKII活性有效磷酸化。对体内和体外32P标记的P蛋白的磷酸肽分析比较表明，这两种蛋白具有共同的磷酸化位点。为了确定CKII介导的磷酸化的确切位点，我们通过定点诱变将位于CKII共有基序内的特定丝氨酸残基改变为丙氨酸。结果表明，位于P蛋白N端一半的Ser 86、Ser 151和Ser 180参与了CKII介导的P蛋白磷酸化。
