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A camelid nanobody against EGFR was easily obtained through refolding of inclusion body expressed in Escherichia coli
Author(s) -
Xu Li,
Song Xiaoyu,
Jia Lingyun
Publication year - 2017
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1544
Subject(s) - escherichia coli , chemistry , inclusion bodies , epidermal growth factor receptor , urea , glutathione , dilution , epidermal growth factor , antibody , biochemistry , a431 cells , microbiology and biotechnology , chromatography , receptor , cell , biology , immunology , gene , enzyme , physics , thermodynamics , molecular medicine , cell cycle
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.