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目的:利用大肠杆菌表达体系制备带有胶原结合结构域(CBD)的骨形态发生蛋白2(BMP2),研究CBD-BMP2表达、纯化及复性的条件和方法。方法:将具有胶原结合能力的CBD基因序列克隆入BMP2基因序列的N端,构建重组蛋白表达质粒pet21b/CBD-BMP2,转化入工程性大肠杆菌BL21菌株内;37℃条件下添加诱导剂异丙基硫代半乳糖苷(IPTG)持续诱导表达;采用Ni-NTA亲和层析柱进行纯化;运用超纯水稀释复性法对纯化后的CBD-BMP2进行复性;0.22μm微孔滤膜对复性后蛋白除菌,通过除菌前后蛋白浓度比值计算回收率;聚丙烯酰胺凝胶电泳(SDS-PAGE)检测重组蛋白表达、纯化以及复性;BCA蛋白定量法测定蛋白浓度。结果:重组质粒pet21b/CBD-BMP2在工程性大肠杆菌中得到充分表达;CBD-BMP2以包涵体形式表达;SDS-PAGE分析,8mol·L~(-1)尿素存在条件下目的蛋白溶解于裂解液上清中,经纯化后目的蛋白单体存在于洗脱液B中,单体相对分子质量约为14 000;稀释复性后SDS-PAGE分析,相对分子质量14 000及28 000处可见2条清晰条带,重组蛋白单链成功复性为二聚体结构,相对分子质量约为28 000;过滤除菌前后目的蛋白浓度分别为110和80mg·L~(-1),回收率约为73%。结论:重组CBD-BMP2载体成功转化至大肠杆菌内,CBD-BMP2蛋白得到了高效的表达和复性。建立了利用原核表达体系制备重组CBD-BMP2蛋白的实验方法。
OBJECTIVE: To prepare bone morphogenetic protein 2 (BMP2) with collagen-binding domain (CBD) by using E. coli expression system and to study the conditions and methods of expression, purification and renaturation of CBD-BMP2. Methods: The CBD gene with collagen binding ability was cloned into the N terminus of BMP2 gene sequence to construct the recombinant protein expression plasmid pet21b / CBD-BMP2 and transformed into E. coli strain BL21. Under the condition of 37 ℃, The recombinant plasmid was purified by Ni-NTA affinity chromatography. The purified CBD-BMP2 was refolded by ultrafiltration with ultrapure water. The 0.22 μm microporous membrane The protein after refolding was sterilized, and the recovery rate was calculated by the ratio of protein concentration before and after sterilization. The protein expression, purification and refolding were detected by SDS-PAGE. The protein concentration was determined by BCA protein quantification. Results: The recombinant plasmid pet21b / CBD-BMP2 was fully expressed in E. coli; CBD-BMP2 was expressed in inclusion bodies; SDS-PAGE analysis showed that the target protein was dissolved in the presence of 8mol·L -1 urea In the liquid supernatant, the purified target protein monomer was present in eluent B. The relative molecular weight of the monomer was about 14 000. SDS-PAGE analysis showed that the relative molecular masses were 14,000 and 28,000 The bands of the recombinant protein were successfully renaturated to dimer structure with molecular weight of about 28 000. The concentrations of target proteins before and after filtration sterilization were 110 and 80 mg · L -1, 73%. CONCLUSION: The recombinant CBD-BMP2 vector is successfully transformed into E. coli. The expression of CBD-BMP2 protein is highly efficient and renaturation. The experimental method for preparing recombinant CBD-BMP2 protein by using prokaryotic expression system was established.