rhBMP-6的表达和纯化  

EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN BMP-6 FUSION PROTEIN E.COLI

作　　者：阳菊华[1] 杨爽[1] 赵丽[1] 罗绍光[1] 张杰[1] 秦刚[1] 朱天慧[1] 

机构地区：[1]南开大学医学院,天津300071

出　　处：《南开大学学报（自然科学版）》2003年第1期91-96,共6页Acta Scientiarum Naturalium Universitatis Nankaiensis

基　　金：天津市自然科学基金资助项目 (0 1 3 60 891 1 )

摘　　要：骨形态发生蛋白 (bonemorphogenetic protein,BMP) -6属于转化生长因子 (transforming growth fac-tor) -β超基因家族 ,在骨的形成中具有重要作用 ,并受雌激素选择性上调 .本研究利用 RT-PCR从人胎盘组织中扩增 BMP-6成熟肽的 DNA片段 ,克隆到 p GEX-5 X-1中 ,构建 GST-BMP6融合蛋白表达载体 p GEX-BMP-6,转化 E.coli DH5 α.克隆质粒转化的工程菌 ,经 IPTG诱导 4h后 ,高效表达 GST-BMP6融合蛋白 ,在 SDS-PAGE上出现一新蛋白带 ,分子量约为 42 k D,约占总蛋白的 2 5 % ,表达产物以包涵体形式存在 .菌体超声破碎 ,经 0 .3 %N-十二烷基肌氨酸钠 (SKL)溶解包涵体 ,离心后的上清液加入 0 .6% Triton X-1 0 0螯合 SKL.然后利用谷胱甘肽 Sepharose-4B亲和层析纯化 .结合 GST-BMP6的介质经洗涤、Xa因子酶切得到纯度达 95 %的 rh BMP-6蛋白 .Bone morphogenetic protein (BMP)-6 is a member of the transforming growth factor-β superfamily and plays a pivotal role in endochodral bone formation and is also an estrogen-selective regulated gene among BMPs. In the present study, the cDNA encoding human BMP-6 mature peptide was amplified by RT-PCR from human placental mRNA and was subcloned into pGEX-5X-1 vector, then generated expression plasmid of GST-BMP-6 fusion protein, pGEX-BMP-6. GST-BMP-6 fusion protein was highly expressed in E.Coli DH5 α after transformation and IPTG induction for 4, and then was purified through single-step affinity chromatography on bulk Glutathione Sepharose-4B from crude sarkosyl solubilized bacterial lysate. The highly-purity rhBMP-6 protein was obtained by removing GST from factor Xa cleavaged fusion protein.

关 键 词：骨形态发生蛋白-6 蛋白纯化 大肠杆菌 rhBMP-6 蛋白表达 转化生长因子 骨形成 

分 类 号：Q510.3[生物学—生物化学]