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蛋白激酶Cα相互作用蛋白(PICK1)的PDZ结构域介导了PICK1和AMPAR的亚基GluR2的C末端结合,在GluR2下调引起兴奋性毒性而产生的多种神经性疾病中,PICK1的PDZ结构域是一个潜在的药物靶标.文章将重组质粒pET3a-pick1和pET-3C-GB-pdz转入E.coli BL21(DE3),经IPTG诱导,在大肠杆菌中获得重组表达,目的蛋白经硫酸铵沉淀或Ni 2+-NTA Sepharose 4Bcolumn亲和层析,再经Sephacryl S-200凝胶层析纯化得到了PICK1和GB-PDZ蛋白.利用荧光光谱法初步分析了款冬提取物与PICK1及其不同结构域的结合情况,结果表明款冬提取物与PICK1的结合位点位于PDZ结构域,款冬提取物能有效抑制PDZ结构域与GluR2的相互作用.
The PDZ domain of protein kinase Cα interacting protein (PICK1) mediates the C-terminal binding of GluR2, a subunit of PICK1 and AMPAR, and in a number of neurological disorders resulting from the down-regulation of GluR2 by excitotoxicity, the PDZ domain of PICK1 Is a potential drug target.The recombinant plasmids pET3a-pick1 and pET-3C-GB-pdz were transformed into E.coli BL21 (DE3), induced by IPTG and recombinantly expressed in E. coli. The target protein was precipitated by ammonium sulfate Or Ni 2 + -NTA Sepharose 4Bcolumn affinity chromatography, and then purified by Sephacryl S-200 gel chromatography to get PICK1 and GB-PDZ protein.Using fluorescence spectroscopy to preliminary analysis of the colchicine extract and PICK1 and its different domains The results showed that the colchicine-PICK1 binding site is located in the PDZ domain, coltsfoot extract can effectively inhibit PDZ domain and GluR2 interaction.