HEPARIN-BINDING EGF–LIKE GROWTH FACTOR AS A PROSPECTIVE MEDIATOR OF TISSUE REPAIR AND REGENERATION

Abstract

Aim. To obtain bioactive recombinant human HB-EGF and investigate its proliferation capacity. Methods. The pET32(a)-HB-EGF cloning plasmid was extracted from E. coli DH10B cells and transformed into E. coli BL21 (DE3) Rosetta cells. The expression of HB-EGF was induced with IPTG and the protein was purified by metal-affinity chromatography with Co²⁺-NTA. Purified HB-EGF was analyzed by SDS-PAGE. The interaction of HB-EGF with receptors was analyzed by flow cytometry using A431 cells. The effect of HB-EGF on the proliferation of 3T3 and L929 fibroblast cell lines was accessed using the MTT assay. Results. High-quality HB-EGF was obtained through an optimized protocol involving plasmid purification, E. coli transformation, and protein purification. SDS-PAGE confirmed the purity of HB-EGF. Flow cytometry validated the binding of EGFP-HB-EGF to A431 cell receptors. The MTT assay data showed that HB-EGF significantly promoted the proliferation of 3T3 cells in a dose-dependent manner, with a 67% and 112% increase in cell density in the presence of 500 ng/mL and 1000 ng/mL of HB-EGF, respectively. No significant changes in the proliferation of L929 cells were observed under the same concentrations of HB-EGF. Conclusions. We have successfully optimized a protocol to produce and purify biologically active HB-EGF in E. coli. The bioactivity of HB-EGF was validated in A431 and 3T3 cell lines, demonstrating its potential to interact with receptors and significantly increase cell proliferation. This bioactive HB-EGF can be potentially used for tissue repair and regeneration.

Keywords

• hb-egf
• recombinant protein
• proliferation
• tissue repair.