重组人胶原绑定骨形态发生蛋白2在大肠杆菌中的表达、纯化与复性

摘要

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 pet21 b／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，8 mol·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14000；稀释复性后SDS-PAGE分析，相对分子质量14000及28000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28000；过滤除菌前后目的蛋白浓度分别为110和80 mg·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 construct the Escherichia coli (E. coli)expression system for preparation of the bone morphogenetic protein-2 (BMP2)with collagen-binding domain (CBD),and to study the methods and conditions for expression, purification and renaturation of CBD-BMP2.Methods:CBD sequence was cloned into the N-terminal of BMP2 sequence, the recombinant vector pet21b/CBD-BMP2 was constructed and transformed into E.coli BL21.The expression of recombinant protein was induced using isopropylβ-D-thiogalactopyranoside (IPTG) at 37 ℃.Ni-NTA chelate chromatography was used to purify CBD-BMP-2.Denaturing CBD-BMP2 was refolded by dilution method using ultrapure water.The refolding CBD-BMP2 was filtered through a 0.22μm microfiltration membrane for degermation.The recovery rate was calculated by the ratio of the protein concentration before and after degermation. The expression, purification, and renaturation of recombinant protein were detected by SDS-PAGE method.The concentration of CBD-BMP2 was detected by BCA assay.Results:The recombinant vector pet21b/CBD-BMP2 was successfully transformed into E.coli BL21,and the recombinant protein was expressed as inclusion bodies in E.coli.The SDS-PAGE results showed denaturing protein was dissolved in supernatant of lysis buffer with 8 mol·L-1 urea and the purified recombinant protein existed in elution buffer B with relative molecular mass about 14 000.Two bands (14 000 and 28 000)were seen in the SDS-PAGE picture,which indicated that the monomer was successfully refolded into dimer by dilution method.The concentrations of recombinant protein before and after degermation were 110 and 80 mg · L-1 , respectively, and the recovery rate was about 73%. Conclusion:The recombinant vector pet21b/CBD-BMP2 is transformed into E.coli BL21 successfully,and the recombinant CBD-BMP2 is expressed and refolded efficiently. The methods of prokaryotic expression system for preparing recombinant CBD-BMP2 protein are established.