国家高技术研究发展计划(863)资助项目(2003AA624150)
This work was supported by a grant from National Hi-Tech Research and Development Program of China (2003AA624150)
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 α1B subunit of N-type voltage-gated calcium channel of Rattus 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.
周玉娟,谢丽萍,张荣庆.大鼠N型钙通道ⅢP区原核表达、纯化及活性研究[J].生物化学与生物物理进展,2009,36(12):1569-1577
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