徐丽

个人信息Personal Information

副教授

硕士生导师

性别:女

毕业院校:协和医科大学

学位:博士

所在单位:生物工程学院

学科:生物化学与分子生物学. 生物化工. 生物医学工程

办公地点:生物楼302

联系方式:13478968672 0411-84706316

电子邮箱:xu-li@dlut.edu.cn

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A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli

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论文类型:期刊论文

发表时间:2017-11-01

发表刊物:BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY

收录刊物:SCIE、EI、PubMed、Scopus

卷号:64

期号:6

页面范围:895-901

ISSN号:0885-4513

关键字:Arg; dilution refolding; EGFR; glutathione; inclusion body; nanobody

摘要:Using anti-EGFR (epidermal growth factor receptor) nanobody is a good choice for diagnoses and therapeutics for high EGFR expression diseases. In the present study, the percentage composition of anti-EGFR nanobody attained 25% of the total cell protein expressed in Escherichia coli BL21 (DE3). However, almost all nanobodies were expressed as inclusion bodies. To acquire active nanobodies, a series of dilution refolding procedures were optimized after inclusion bodies were dissolved into 6 M urea and purified with immobilized metal affinity chromatography. The results showed the refolding rate of the anti-EGFR nanobodies attained to 73%, and about 100 mg nanobodies were refolded from 1 L cells under the conditions that the initial nanobody concentration was 0.3 mg/mL, the dilution speed was 2.5 mL/Min, the dilution buffer was Tris-HCl at pH 8.0, the additives were 0.2 M Arg, 5 mM reduced glutathione (GSH), and 1 mM oxidized glutathione (GSSG). Then the activity of the refolded nanobodies was confirmed. The results showed that the refolded anti-EGFR nanobodies, in a dose-dependent manner, bounded to the tumor cell surface of A431 and MCF-7 and significantly inhibited the proliferation of A431 caused by the epidermal growth factor. Our study provides a facile method to rapidly, efficiently, and massively prepare anti-EGFR antibodies and promotes anti-EGFR-based recognition in cancer diagnoses and therapeutics. (C) 2016 International Union of Biochemistry and Molecular Biology, Inc.