Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase.
Construction of an epitope-tagged calmodulin useful for the analysis of calmodulin-binding proteins: addition of a hemagglutinin epitope does not affect calmodulin-dependent activation of smooth muscle myosin light chain kinase.
复制标题
用于分析钙调蛋白结合蛋白的表位标记的钙调蛋白的构建：添加血凝素表位不会影响平滑肌肌球蛋白轻链激酶的钙调蛋白依赖性激活。
DOI：
10.1006/abio.1997.2319
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
O'Connor,CM
中科院分区：
文献类型：
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作者：
Szymanska,G;O'Connor,MB;O'Connor,CM
表位标记的钙调素（CaM），能够与细胞提取物中的钙调素结合蛋白相互作用，将是一个有价值的工具，用于识别蛋白质的信号转导通路涉及钙。通过将流感病毒血凝素（HA）蛋白的9个氨基酸部分的编码序列插入鸡CaM的过表达载体的起始位点，构建了表位标记的CaM的细菌过表达载体。将细菌中产生的HA-CaM融合物与天然CaM进行比较，以确定其激活平滑肌肌球蛋白轻链激酶（MLCK）的能力，MLCK是最了解的CaM依赖性酶之一。MLCK活性在纯化的系统和CaM耗尽的“天然肌动球蛋白”制剂中进行了测试，该制剂保持了完整平滑肌的许多调节特性。HA-CaM在两个系统中的行为与未修饰的CaM相同，表明HA表位不会对CaM功能产生不利影响。重组HA-CaM用于灵敏地检测钙调素与平滑肌蛋白的相互作用，在一个修改的凝胶覆盖试验，使用单克隆抗体对HA表位作为第二试剂。HA-CaM和MLCK的酶活性复合物可以使用相同的单克隆抗体和蛋白G-Sepharose珠从肌动球蛋白制剂中免疫沉淀。
An epitope-tagged calmodulin (CaM), capable of interacting with CaM-binding proteins in cellular extracts, would be a valuable tool for identifying proteins in signal transduction pathways involving calcium. A bacterial overexpression vector for epitope-tagged CaM has been constructed by inserting the coding sequence for a nine amino acid portion of the influenza virus hemagglutinin (HA) protein into the initiation site of an overexpression vector for chicken CaM. The HA–CaM fusion produced in bacteria was compared to native CaM for its ability to activate smooth muscle myosin light chain kinase (MLCK), one of the best understood CaM-dependent enzymes. MLCK activity was tested in both a purified system and a CaM-depleted “native actomyosin” preparation maintaining many of the regulatory properties of the intact smooth muscle. HA–CaM behaves identically to unmodified CaM in both systems, indicating that the HA epitope does not adversely affect CaM function. The recombinant HA–CaM was used to sensitively detect CaM interactions with smooth muscle proteins in a modified gel overlay assay, using a monoclonal antibody against the HA epitope as the secondary reagent. Enzymatically active complexes of HA–CaM and MLCK could be immunoprecipitated from actomyosin preparations using the same monoclonal antibody and protein G–Sepharose beads.