title: Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR.
abstract: The epidermal growth factor receptor (EGFR), overexpressed in many epithelial tumors, is emerging as an attractive target for cancer therapy. Antibodies to the extracellular region of EGFR play a key role in the development of a mechanistic understanding and cancer therapy. In the present study, we demonstrated for the first time that EGFR-truncated extracellular domain (EGFR-tED), which was expressed in Escherichia coli BL21 (DE3) cells in the form of inclusion bodies, could be purified and renatured. The EGFR-tED protein was purified by gel filtration and Ni-NTA affinity chromatography with high purity (>90%) and refolded by a urea gradient size-exclusion chromatography, which could bind its ligand EGF in a concentration-dependent manner. The renatured EGFR was used for biopanning anti-EGFR scFvs from a human synthetic antibody phage display library. Combined with an additional cell-based ELISA screen, a novel scFv, E10, was obtained with two-fold more potent on the binding to EGFR-bearing tumor cells (the epidermoid carcinoma cell line A431) and the inhibition of A431 cells proliferation than scFv 11F8, suggesting that the E10 has the potential to be developed as therapeutic agents to solid tumors associated with EGFR overexpression.
authors: State Key Laboratory of Natural Medicines (China Pharmaceutical University), School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China.
authors: Yaqiong
authors: Y
authors: Zhou
authors: Juan
authors: J
authors: Zhang
authors: Haizhen
authors: H
authors: Jin
authors: Zhiguo
authors: Z
authors: Chen
authors: Qinhang
authors: Q
authors: Wu
authors: Weiguang
authors: W
authors: Li
authors: Ming
authors: M
authors: Yue
authors: Chen
authors: C
authors: Luo
authors: Min
authors: M
authors: Wang
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011485:Protein Binding; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
chemical_list: D019151:Peptide Library; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors
keywords: Epidermal growth factor receptor (EGFR); Phage display; Prokaryotic expression; Refolding; Single-chain variable fragment antibody (scFv); Tumor therapy