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

doi:10.13481/j.1671-587x.20160207

Abstract

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 isopropyl β-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 0.22 μm 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 14000.Two bands (14000 and 28000) 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.

Key words: collagen binding domain, bone morphogenetic protein-2, inclusion body, renaturation

CLC Number:

WU Naipeng, WANG Yu, SONG Jia, WU Zhenxu, GAO Tianlin, FENG Xiangru, FU Chuan, WANG Zongliang, WANG Chunyan. Expression,purification and renaturation of recombinant human collagen-binding bone morphogenetic protein-2 from Escherichia coli[J].Journal of Jilin University Medicine Edition, 2016, 42(02): 226-230.

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Expression,purification and renaturation of recombinant human collagen-binding bone morphogenetic protein-2 from Escherichia coli

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