INTERNATIONAL CONGRESS OF
CancerGenetics
Engineering and Production of a Recombinant HER3-Binding Affibody-EGFP Fusion Protein as a Potential Diagnostic Probe for Cancer Research
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Sahar Babaei Khorzoughi,1,* Mojtaba Mortazavi,2 Rahman Emamzadeh,3
1. Graduate University of Advanced Technology
2. Graduate University of Advanced Technology
3. University of Isfahan
- Introduction: HER3 (ErbB3) is a member of the receptor tyrosine kinase family that plays a key role in cancer progression and therapy resistance, especially in breast cancer. Although its intrinsic kinase activity is weak, HER3 can form heterodimers with HER2 or other ErbB receptors, triggering potent signaling pathways. Overexpression of HER3 is linked to poor prognosis and decreased response to targeted therapies, making it an important biomarker for diagnosis and therapeutic monitoring. Traditional antibody-based methods have been widely used to detect HER3, but they often come with drawbacks such as high cost, large size, and slower tissue penetration. Affibodies, on the other hand, are small engineered proteins (~7 kDa) with high stability and binding specificity. When fused to a fluorescent reporter like enhanced green fluorescent protein (EGFP), they can serve as reliable biosensors for detecting tumor markers in a rapid and cost-effective way. In this work, we designed and produced a recombinant fusion protein combining the HER3-binding affibody Z08699 with EGFP. Our goal was to develop a stable, functional, and easy-to-produce probe that could be used as a molecular tool for cancer research and potentially for diagnostic purposes.
- Methods: The gene encoding the HER3-binding affibody Z08699 was fused in-frame to EGFP and cloned into the pET-21a(+) bacterial expression vector. A C-terminal His-tag was included to facilitate purification. The construct was transformed into E. coli BL21(DE3) cells. For protein expression, cultures were grown in LB medium with ampicillin at 37°C until mid-log phase and then induced with 1 mM IPTG. We optimized induction conditions, varying temperature and duration to maximize the soluble fraction. Cells were harvested, lysed by sonication, and the recombinant protein was purified under native conditions using Ni-NTA affinity chromatography. Purity and molecular weight were checked by SDS-PAGE, while protein concentration was measured using the Bradford assay. Fluorescence emission spectra were recorded to confirm proper folding and activity of the EGFP domain after fusion.
- Results: Successful cloning of the affibody-EGFP construct was confirmed by restriction digestion and sequencing. Following IPTG induction, a clear band at the expected molecular weight (~34 kDa) appeared on SDS-PAGE, confirming protein expression. Inducing at 25°C for 16 hours increased the solubility of the fusion protein compared to higher temperatures. Ni-NTA purification produced a highly enriched protein fraction with minimal contaminants. Fluorescence analysis showed a strong peak at ~510 nm, indicating that EGFP retained its proper folding and activity. The recombinant protein was stable and reproducible across multiple batches, making it suitable for downstream applications.
- Conclusion: We successfully engineered, expressed, and purified a HER3-binding affibody fused to EGFP. The fusion protein retained both high purity and fluorescence activity, demonstrating its potential as a robust diagnostic probe. Compared to conventional antibodies, affibody-based probes offer advantages in size, production speed, and cost, while maintaining high specificity and stability. This construct could serve as a reliable tool for HER3 detection in cancer research, with possible applications in early diagnosis, therapeutic monitoring, and research on targeted therapies. Future work will focus on testing the probe in various HER3-expressing cancer cell lines and exploring its integration into biosensing platforms or imaging systems, potentially expanding its use in translational oncology.
- Keywords: HER3, Affibody, EGFP, Recombinant protein, Cancer cells
