大鼠N型钙通道ⅢP区原核表达、纯化及活性研究(英文)  

Expression and Purification of The Third P Region of α1B Subunit of N-type Calcium Channel of Rattus norvegicus

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作  者:周玉娟[1] 谢莉萍[1,2] 张荣庆[1,2] 

机构地区:[1]清华大学生物科学与技术系,北京100084 [2]清华大学教育部蛋白质科学实验室,北京100084

出  处:《生物化学与生物物理进展》2009年第12期1569-1577,共9页Progress In Biochemistry and Biophysics

基  金:supported by a grant from National Hi-Tech Research and Development Program of China (2003AA624150)~~

摘  要:高保真PCR克隆出编码大鼠海马组织N型钙通道α1B亚基ⅢP区的DNA序列,分别与表达载体pET28b和pGEX-4T-1连接,转化入能补充稀有密码子tRNAs的大肠杆菌Rosetta菌株中,通过优化表达条件,实现了高效诱导表达.再通过低温诱导,将包涵体用尿素溶解再稀释后透析法复性、酶切、亲和柱层析等方法得到较纯的可溶性目的蛋白HIS-Cav22P、GST-Cav22P和Cav22P,紫外吸收光谱检测实验证明了重组蛋白HIS-Cav22P能和钙离子可逆地结合.最后通过GST沉降实验证明了芋螺毒素SO3与Cav22P存在体外相互结合作用.上述结果为揭示芋螺毒素SO3特异性阻断大鼠海马组织N型钙流的分子机制提供了直接的依据,也为建立筛选新的非吗啡型天然镇痛药物的技术平台提供了基础.The DNA fragment coding the third P region of α 1B subunit of N-type voltage-gated calcium channel ofRattus norvegicus (Cav22P for short) was amplified by high fidelity PCR, inserted into vectors pET28b and pGEX-4T-1 respectively, and expressed in Escherichia coli Rosetta. The expressed product HIS-Cav22P mostly deposited in an inclusive body. The inclusive body of HIS-Cav22P dissolved in urea buffer. After dilution and dialysis, the refolded protein HIS-Cav22P was finally purified via Histrap chelating HP column. Ultraviolet spectroscopy results demonstrated that HIS-Cav22P protein can bind to calcium ions reversely. The expressed product GST-Cav22P was purified from the supernatant of cell lysate using Glutathione Sepharose 4B column. However, GST-Cav22P degraded severely, which caused it difficult to purify the protein Cav22P and the GST pull-down assay. All the results suggest that the active recombinant proteins of Cav22P may act as a molecular target for high through screen of non-narcotic analgesic drugs.

关 键 词:纯化 表达 N型钙通道 

分 类 号:Q78[生物学—分子生物学]

 

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