重组人BMP6在大肠杆菌中可溶表达、纯化及活性分析
Soluble Expression of Recombinant Human BMP6 in Escherichia coli and Its Purification and Bioassay in Vitro
投稿时间:2007-06-26  
DOI:  
中文关键词:BMP6, MBP, BL21 trxB(DE3), 可溶性
英文关键词:BMP6, MBP, BL21 trxB(DE3), solubility
基金项目:天津市自然科学基金资助 (No. 05YFJMJC01800)。
作者单位E-mail
雷荣悦 南开大学医学院分子遗传学实验室, 天津 300071  
乔玉欢 南开大学医学院分子遗传学实验室, 天津 300071  
闫继东 南开大学医学院分子遗传学实验室, 天津 300071  
杨爽 南开大学医学院分子遗传学实验室, 天津 300071  
朱天慧 南开大学医学院分子遗传学实验室, 天津 300071 zhuth@nankai.edu.cn 
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中文摘要:
      BMP6是一种调节成骨细胞和成软骨细胞分化的骨诱导因子, 在修复各种骨缺损方面具有很好的应用潜力。有诱骨活性的BMP6是多二硫键的二聚体蛋白, 疏水性极强容易聚集沉淀。为了在大肠杆菌中可溶表达具有生物活性的重组人BMP6(rhBMP6), 构建了具有TRX、GST、MBP、CBD融合标签和His6标签的 rhBMP6成熟肽原核表达载体, 调节诱导温度和IPTG浓度, 比较不同融合标签和诱导条件对目的蛋白表达量和溶解性的影响。结果表明, MBP最能有效的增强rhBMP6的溶解性, 诱导条件对溶解性影响较小。大肠杆菌BL21 trxB(DE3)这种硫氧还蛋白还原酶缺陷菌株为rhBMP6二硫键在胞质中形成提供了合适的氧化还原环境。MBP和BL21 trxB(DE3)相结合在细胞质中高效可溶表达出了BMP6融合蛋白二聚体。表达产物经亲和层析和凝胶排阻层析纯化后, 能诱导成肌细胞系C2C12向成骨细胞方向 转化。
英文摘要:
      BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.
雷荣悦,乔玉欢,闫继东,杨爽,朱天慧.重组人BMP6在大肠杆菌中可溶表达、纯化及活性分析[J].生物工程学报,2008,24(3):452~459
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