重组人骨形态发生蛋白-6的表达、纯化及其活性分析  

Expression of Recombinant Human BMP_6 in Escherichia coli and Its Purification and Bioassay in vitro

作　　者：阳菊华[1] 赵丽[1] 杨爽[1] 吴双庆[1] 张杰[1] 朱天慧[1] 

机构地区：[1]南开大学医学院医学分子遗传实验室,天津300071

出　　处：《生物工程学报》2003年第5期556-560,共5页Chinese Journal of Biotechnology

基　　金：天津市自然科学基金资助 (No.0 13 60 8911)~~

摘　　要：利用RT_PCR从人胎盘组织中获取BMP_6成熟肽的cDNA片段 ,并克隆到表达载体pET_15b中 ,构建hBMP_6成熟肽的非融合蛋白表达质粒pET_BMP6 ,转化E .coliBL2 1(DE3)。IPTG诱导 4h后 ,工程菌高表达rhBMP_6成熟肽 ,在SDS_PAGE上出现预期的新蛋白带 (≈ 15kD) ,约占菌体总蛋白的 10 % ,表达产物以包涵体形式存在。分离和纯化的包涵体溶解于 8mol L尿素 ,在变性溶解状态下经阳离子交换层析 ,得到目的蛋白纯度达 95 %以上。再经稀释复性后 ,约 80 %的rhBMP_6形成同源二聚体。体外活性分析结果显示 :rhBMP_6可以提高C3H10T1 2细胞碱性磷酸酶活性及促进I型胶原、Osterix(Osx)和骨钙素 (Osteocalcin)等成骨细胞表型转化标记基因mRNA的表达 。To purify the recombinant human BMP_6 protein and to establish its in vitro bioassay method. The cDNA encoding the mature peptide of hBMP_6 protein was amplified by reverse transcription_polymerase chain reaction (RT_PCR), using human placental mRNA as template, and subcloned into the high_expression vector pET_15b under the control of T7 lac promoter. The resulting construct, pET_BMP6, was then transformed into an Escherichia coli strain BL21 (DE3) for the production of recombinant hBMP_6 protein (rhBMP_6). After 4 hours of induction by isopropyl_β_D_ thiogalactoside (IPTG), rhBMP_6 (≈15kD) was expressed and formed inclusion bodies, contributing up to 10% of the total bacterial protein. The inclusion bodies were isolated and redissolved in 8mol/L urea, and the denatured rhBMP_6 was purified to 95% purity by CM_Cellulose 32 ion exchange chromatography (IEC). The osteoinductivity of rhBMP_6 was measured by the expression of some of the osteoblast differentiation marker genes in rhBMP_6_treated C3H10T1/2 cells as reflected by determinations of alkaline phosphatase (ALPase) activity and semi_quantitative RT_PCR. At the end of the purification process, about 80% of rhBMP_6 formed disulphide_linked homodimers after refolding during renaturation. The apparent size of the protein was 30kD on non_reducing SDS_PAGE, similar to that of the native form of hBMP_6 The enzyme assays showed that the ALPase activity was increased in a dose_dependent manner with the treatment of rhBMP_6 After the addition of 300ng/mL of rhBMP_6, the ALPase activity of C3H10T1/2 cells increased significantly. The activity of rhBMP_6 used was comparable to about 70% of that of the standard hBMP_6 derived from eukaryotic cells. RNA extraction data also showed rhBMP_6 stimulated expression of osteoblast marker genes, including type I collagen, osterix, and osteocalcin in a time_dependent manner. After 5 days of treatment, their level of expression was increased to 3 times that of controls. Bone morphogenetic protein (BMP)_6, a membe

关 键 词：重组 人骨形态发生蛋白-6 表达 纯化 活性 

分 类 号：Q786[生物学—分子生物学]