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Updated: May 11, 2026
Screening and Identification of Small Peptides Targeting Fibroblast Growth Factor Receptor2 using a Phage Display Peptide Library
Published on: September 30, 2019
Prokaryotic expression and refolding of EGFR extracellular domain and generation of phage display human scFv against
Yaqiong Zhou1, Juan Zhang, Haizhen Jin
1State Key Laboratory of Natural Medicines (China Pharmaceutical University), School of Life Science & Technology, China Pharmaceutical University, Nanjing 210009, PR China.
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.
Insights
Researchers successfully purified and renatured a truncated extracellular domain of the epidermal growth factor receptor (EGFR). This enabled the development of a novel antibody, E10, showing potent anti-cancer activity against EGFR-overexpressing tumor cells.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- Epidermal growth factor receptor (EGFR) is overexpressed in many epithelial tumors, making it a key target for cancer therapy.
- Antibodies targeting the extracellular region of EGFR are crucial for understanding its role in cancer and developing treatments.
Purpose of the Study:
- To demonstrate the purification and refolding of a truncated extracellular domain of EGFR (EGFR-tED).
- To develop novel anti-EGFR single-chain variable fragments (scFvs) for potential cancer therapeutics.
Main Methods:
- EGFR-tED was expressed in E. coli, purified using gel filtration and Ni-NTA affinity chromatography, and refolded via urea gradient size-exclusion chromatography.
- The refolded EGFR-tED was used for biopanning a synthetic antibody phage display library to isolate anti-EGFR scFvs.
- A novel scFv, E10, was identified through biopanning and cell-based ELISA, demonstrating enhanced binding and inhibitory effects.
Main Results:
- Highly pure EGFR-tED (>90%) was successfully purified and renatured, retaining its ability to bind EGF.
- A novel anti-EGFR scFv, E10, was isolated, exhibiting a two-fold greater potency in binding to EGFR-expressing A431 cells compared to scFv 11F8.
- E10 demonstrated superior inhibition of A431 cell proliferation compared to scFv 11F8.
Conclusions:
- The study successfully produced a functional, renatured EGFR-tED, facilitating the development of targeted cancer therapies.
- The novel scFv E10 shows significant potential as a therapeutic agent for solid tumors characterized by EGFR overexpression.
