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

吉林大学学报(医学版) ›› 2016, Vol. 42 ›› Issue (02): 226-230.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

吴乃蓬¹, 王宇², 宋佳¹, 武振旭², 高田林², 冯祥汝², 付川², 王宗良², 王春艳¹

1. 吉林大学公共卫生学院卫生检验学教研室, 吉林长春 130021;
2. 中国科学院长春应用化学研究所再生医学材料课题组, 吉林长春 130021

收稿日期:2015-11-11 发布日期:2016-03-31
通讯作者: 王春艳,副教授,硕士研究生导师(Tel:0431-85619441,E-mail:chunyan@jlu.edu.cn) E-mail:chunyan@jlu.edu.cn
作者简介:吴乃蓬(1989-),男,山东省蓬莱市人,在读医学硕士,主要从事重组蛋白制备以及应用方面的研究。
基金资助:
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(20130413059GH)

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

WU Naipeng¹, WANG Yu², SONG Jia¹, WU Zhenxu², GAO Tianlin², FENG Xiangru², FU Chuan², WANG Zongliang², WANG Chunyan¹

1. Department of Health Laboratory, School of Public Health, Jilin University, Changchun 130021, China;
2. Regenerative Medicine Material Research Group, Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130021, China

Received:2015-11-11 Published:2016-03-31

摘要/Abstract

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;0.22 μm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,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。

关键词: 胶原结合结构域, 骨形态发生蛋白2, 包涵体, 复性

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

中图分类号:

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(02): 226-230.

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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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

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