CHARACTERIZATION OF THE HUMAN CALMODULIN-LIKE PROTEIN EXPRESSED IN ESCHERICHIA-COLI
CHARACTERIZATION OF THE HUMAN CALMODULIN-LIKE PROTEIN EXPRESSED IN ESCHERICHIA-COLI
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DOI：
10.1021/bi00166a017
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发表时间：
1992-12-29
期刊：
影响因子：
2.9
通讯作者：
STREHLER, EE
中科院分区：
文献类型：
--
作者：
RHYNER, JA;KOLLER, M;STREHLER, EE
无内含子的人钙调素样基因的蛋白质编码区[Koller，M.，& Strehler，E. E. 04 The Dog（1988）239，121-128]的重组质粒插入pKK 233 -2表达载体，转化大肠杆菌JM 105，经苯基-琼脂糖亲和层析纯化，得到148个残基，M（r）= 16 800的人蛋白。从1 L细菌培养物中获得几毫克纯化蛋白。人CLP的一些特性进行了比较，细菌表达的人钙调素（CaM）和牛脑钙调素。CLP显示了一个特征的钙离子依赖的SDS-聚丙烯酰胺凝胶电泳迁移率的变化，虽然这种变化的幅度小于所观察到的钙调素。CLP能够将3 '，5'-环核苷酸磷酸二酯酶激活到与正常CaM相同的V（max），尽管K（act）高7倍。而红细胞膜Ca ~（2+）-ATP酶活性仅为CLP最大活性的62%。CLP被发现含有4个Ca 2+结合位点，平均亲和常数为10（5）M-1，比在可比条件下的CaM低约10倍。高度组织特异性表达的CLP代表了一种新的人Ca 2+结合蛋白，显示出CaM同种型的特征。
The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16 800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca2+-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same V(max) as normal CaM, albeit with a 7-fold higher K(act). In contrast, the erythrocyte plasma membrane Ca2+-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca2+-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca2+-binding protein showing characteristics of a CaM isoform.