兔Oddi氏括约肌细胞BK通道Alpha亚基在Pichia酵母中的高效表达、纯化和鉴定

目的用巴斯德毕赤酵母系统表达兔Oddi氏括约肌(SO)细胞BK通道α亚基,并进行纯化和鉴定。方法采用RTPCR扩增编码兔SO细胞BK通道α亚基B细胞表位区基因的cDNA,经测序正确将其克隆入酵母表达载体pPIC9K,以电穿孔法转化酵母GS115。经MD平板筛选重组子、G418筛选鉴定后,甲醇诱导表达,镍离子亲合层析法对表达上清进行纯化,进行SDSPAGE和免疫印迹分析。结果用RTPCR扩增出约1497bp的兔SO细胞BK通道α亚基基因的cDNA。序列分析显示,扩增序列与已发布的新西兰大白兔骨骼肌BK通道α亚基的cDNA序列完全一致。SDSPAGE显示本系统表达的α亚基融合蛋白Mr约为55000,表达量约为55mg/L,纯度为96%。Westernblot检测证明,该α亚基融合蛋白可与兔BK通道α亚基多克隆抗体特异性结合。结论克隆了编码兔SO细胞BK通道α亚基B细胞表位区基因的cDNA,并在毕赤酵母中成功地表达了该蛋白,为进一步研究BK通道提供了物质基础。

Expression, purification and identification of Alpha subunit of rabbit SO cell BK channel in methylotrophic yeast Pichia pastoris

Objective To express, purify and identify α subunit of rabbit SO cell BK channel in methylotrophic yeast Pichia pastoris. Methods Gene encoding the B cell epitope regions of α subunit of rabbit SO cell BK channel was amplified by RT-PCR and cloned into the vector pPIC9k.The constructed plasmid was transformed into yeast GS115 by electroporation. The recombinant transformants with a high copy number of the plasmid were selected by using MD plate and G418. The expression of α subunit was induced by addition of methanol. The supernatant was collected and purified with Ni 2+ -NTA resin and analyzed by SDS-PAGE and Western blot. Results A unique band about 1 497bp was amplified by RT-PCR. The amplified sequence was consistent with the published one shown by sequence analysis. The SDS-PAGE analysis showed that the M_r of α subunit product was about 55 000. The concentration of the fusion protein was 55 mg/L and the purity was 96%. The α subunit could be recognized by rabbit multiclonal antibody against α subunit of BK channel, as shown by Western blot. Conclusion The gene encoding the B cell epitope regions of α subunit of rabbit SO cell BK channel has been cloned and expressed successfully in yeast Pichia pastoris, which will play an important role in the further study of BK channel.