人β-微管蛋白2C蛋白的原核表达与纯化

目的构建体外原核表达和纯化人β-微管蛋白2C(TubB2C)蛋白。方法通过聚合酶链反应(PCR)扩增出人TubB2C基因完整开放读码框架(ORF),并插入pGEX-6P-1载体中构建pGEX-TubB2C重组质粒。经大肠杆菌BL21转化后,用异丙醇-β-D-硫代半乳糖苷(IPTG)诱导谷胱甘肽S转移酶(GST)-TubB2C融合蛋白表达,用免疫印迹(W estern b lot)分析鉴定表达的GST-TubB2C蛋白,并在非变性条件下利用G lutath ione Sepharose 4B纯化GST-TubB2C蛋白。结果酶切分析和DNA测序表明TubB2C cDNA完整ORF以正确的阅读框架插入pGEX-6P-1载体;聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示表达的GST-TubB2C蛋白相对分子量约为78 000,W estern b lot结果显示该融合蛋白可被抗GST抗体特异性识别。纯化后的GST-TubB2C蛋白纯度约90%。结论TubB2C蛋白可在体外大量表达和纯化。

Prokaryotic expression and purification of human beta tubulin 2C protein

Objective To build prokaryotic expression in vitro and purify human beta-tubulin 2C （TubB2C） protein. Methods Full open reading frame （ORF） of human TubB2C cDNA was amplified by polymerase chain reaction （PCR） and then was inserted into pGEX-6P-1 carrier to build pGEX-TubB2C recombinant plasmid. After transformed Escheribhia coli BL21, GST-TubB2C proteins was induced by isopropy-β-D-thiogalactoside（IPTG） ,and identified by Western blot and purified using Glutathione Sepharose 4B under non-denaturing conditions. Results Restriction enzyme digestion and direct automated sequencing of DNA demonstrated directional cloning of human TubB2C into the vector pGEX-6P-1 with correct open reading frame. The expression of TubB2C protein with expected molecular weight （about 78 000） was detected by SDS-PAGE and then confirmed by Western Blot. After purification, the purity of GST-TubB2C protein was about 90 percent. Conclusion Human TubB2C protein can be successfully expressed and purified in vitro.