吉林大学学报（医学版）2016，Vol.42Issue(2)：226-230,5.DOI:10.13481/j.1671-587x.20160207
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
Expression,purification and renaturation of recombinant human collagen-binding bone morphogenetic protein-2 from Escherichia coli
摘要
Abstract
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.关键词
胶原结合结构域/骨形态发生蛋白 2/包涵体/复性Key words
collagen binding domain/bone morphogenetic protein-2/inclusion body/renaturation分类
生物科学引用本文复制引用
吴乃蓬,王宇,宋佳,武振旭,高田林,冯祥汝,付川,王宗良,王春艳..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[J].吉林大学学报（医学版）,2016,42(2):226-230,5.基金项目
国家自然科学基金资助课题（51203152）；吉林省科技厅科技发展计划国际合作项目资助课题 （）
