胰岛素受体的配体结合结构域的克隆与表达  

Cloning and Expression of Insulin Receptor Ligand-bindi ng Domains

作　　者：张弘[1] 乔志松[1] 冯佑民[1] 

机构地区：[1]中国科学院上海生命科学研究院,上海生物化学研究所,分子生物学国家重点实验室,上海200031

出　　处：《生物化学与生物物理学报》2000年第6期627-632,共6页

基　　金：国家自然科学基金资助 (No .39670 179)~~

摘　　要：用PCR方法从人胎盘cDNA库中获得编码胰岛素受体α亚基中结合胰岛素的相对独立的结构域L1、L2以及人工设计的L1 (Ala) 10 L2的基因 ,克隆入含T7噬菌体RNA聚合酶启动子的表达质粒 pET 3a中 ,转化大肠杆菌BL2 1(DE3 ) ,用IPTG诱导表达成功。DNA测序、氨基酸组成分析以及蛋白质N端测序证明所表达的蛋白质正确。经过包涵体的分离、洗涤、溶解和纯化 ,得到了纯的变性状态受体的胰岛素高亲和结构域L2、低亲和结构域L1及用 10个丙氨酸相连的两结构域L1 (Ala) 10 L2 ,它们都远小于α亚基且不会由于形成链间二硫键而聚集 ,为进一步研究“胰岛素 受体 (结构域 )”复合物的性质 。Insulin receptor is a transmembrane protein consisting of fo ur subunits, that form a heterotetramer( α 2β 2 )with molecular weight of ～ 350 kD. Because the extracellular subunit(α)consists of 731 residues and a cyst eine rich domain, it is difficult to express and crystallize such a large ligan d binding subunit, thus hampering further study on'insulin receptor'complex. Based on the fact that the domains L1 and L2 of the α subunit, consisted of 1 19 and 118 residues, contained the high and low affinity insulin binding sites, respectively, the cDNAs of L1 and L2 were obtained from a human placental cDNA l ibrary by PCR. The cDNAs of L1, L2 and L1 (Ala) 10 L2 (designed ten a lanine connected L1 and L2) were cloned, respectively, into an expression plas mid pET 3a, and E. coli BL21(DE3)transformants with such plasmids were succ essfully induced to express the goal proteins. The expression products were iso lated and purified by the washing and solubilization of inclusion body, gel filt ration chromatography and ion exchange chromatography. Each final product displ ayed a single band, corresponding the purity above 99%, in SDS PAGE. These pro ducts have also been confirmed respectively as the L1, L2 and L1 (Ala) 10 L2 by DNA sequencing, amino acid composition analysis and N terminal amino ac id sequencing.

关 键 词：胰岛素 受体 胞外结构域 克隆 表达 

分 类 号：Q785[生物学—分子生物学]