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Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR.
Biomed Pharmacother 2013; 67(8):737-43BP

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+Show Affiliations

State Key Laboratory of Natural Medicines (China Pharmaceutical University), School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23639232

Citation

Zhou, Yaqiong, et al. "Prokaryotic Expression and Refolding of EGFR Extracellular Domain and Generation of Phage Display Human scFv Against EGFR." Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, vol. 67, no. 8, 2013, pp. 737-43.
Zhou Y, Zhang J, Jin H, et al. Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomed Pharmacother. 2013;67(8):737-43.
Zhou, Y., Zhang, J., Jin, H., Chen, Z., Wu, Q., Li, W., ... Wang, M. (2013). Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. Biomedicine & Pharmacotherapy = Biomedecine & Pharmacotherapie, 67(8), pp. 737-43. doi:10.1016/j.biopha.2013.03.019.
Zhou Y, et al. Prokaryotic Expression and Refolding of EGFR Extracellular Domain and Generation of Phage Display Human scFv Against EGFR. Biomed Pharmacother. 2013;67(8):737-43. PubMed PMID: 23639232.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against EGFR. AU - Zhou,Yaqiong, AU - Zhang,Juan, AU - Jin,Haizhen, AU - Chen,Zhiguo, AU - Wu,Qinhang, AU - Li,Weiguang, AU - Yue,Ming, AU - Luo,Chen, AU - Wang,Min, Y1 - 2013/04/18/ PY - 2013/03/10/received PY - 2013/03/24/accepted PY - 2013/5/4/entrez PY - 2013/5/4/pubmed PY - 2014/6/27/medline KW - Epidermal growth factor receptor (EGFR) KW - Phage display KW - Prokaryotic expression KW - Refolding KW - Single-chain variable fragment antibody (scFv) KW - Tumor therapy SP - 737 EP - 43 JF - Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie JO - Biomed. Pharmacother. VL - 67 IS - 8 N2 - 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. SN - 1950-6007 UR - https://www.unboundmedicine.com/medline/citation/23639232/Prokaryotic_expression_and_refolding_of_EGFR_extracellular_domain_and_generation_of_phage_display_human_scFv_against_EGFR_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0753-3322(13)00048-6 DB - PRIME DP - Unbound Medicine ER -