Another version of the human insulin receptor kinase domain: expression, purification, and characterization.
Another version of the human insulin receptor kinase domain: expression, purification, and characterization.
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人胰岛素受体激酶结构域的另一个版本：表达、纯化和表征。
DOI：
10.1073/pnas.86.20.7848
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发表时间：
1989
影响因子：
11.1
通讯作者：
Rosen,OM
中科院分区：
文献类型：
--
作者：
Villalba,M;Wente,SR;Russell,DS;Ahn,JC;Reichelderfer,CF;Rosen,OM
我们通过使用杆状病毒表达载体pVL941过表达了另一种胰岛素受体激酶分子，该分子由残基941-1343组成，包括人胰岛素受体。与该表达系统中胰岛素受体激酶的前两种制剂不同，该分子包含人胰岛素受体β亚基胞质结构域的完整未修饰序列。我们的构建体允许在培养的 Sf9 细胞和卷心夜蛾 (Trichoplusia ni) 幼虫中高水平表达重组蛋白。改进的纯化程序可从细胞中以 55% 的产率产生大于或等于 95% 的纯蛋白质。这种纯化蛋白的比活性比之前描述的杆状病毒胰岛素受体激酶（包括来自前受体的残基 946-1343）高 3.5 倍。与后一种分子一样，本文报道的胰岛素受体激酶分子作为单体沉积，其自身磷酸化通过分子内过程发生。提供了有关人胰岛素受体胞质结构域光谱特征的初步数据。
We have overexpressed another insulin receptor kinase molecule, which consists of residues 941-1343 inclusive of the human insulin receptor, by using the baculo-virus expression vector pVL941. Unlike the two previous preparations of insulin receptor kinase in this expression system, this molecule contains the complete unmodified sequence of the cytoplasmic domain of the human insulin receptor beta subunit. Our construct allows high-level expression of the recombinant protein in cultured Sf9 cells and in cabbage looper (Trichoplusia ni) larvae. An improved purification procedure yields greater than or equal to 95% pure protein in 55% yield from the cells. The specific activity of this purified protein is 3.5-fold greater than from the previously described baculovirus insulin receptor kinase (that included residues 946-1343 from the proreceptor). Like the latter molecule, the insulin receptor kinase molecule reported here sediments as a monomer, and its autophosphorylation occurs by an intramolecular process. Preliminary data about the spectroscopic features of the cytosolic domain of the human insulin receptor are presented.