Efficient Expression, Purification and Functional Evaluation of Human Epidermal Growth Factor-Collagen Binding Domain Hybrid Protein on Growth Stimulation of Human Fibroblast Cells

Growth factors such as human epidermal growth factor (hEGF) have recently received high interest in regenerative medicine and pharmaceutical industries mainly due to their ability to restore tissues proliferation and improvement of their biological functions. In spite of various hEGF applications, its efficient expression in Escherichia coli could not yet reach an industrial reality mainly due to the lack of the ability of folding into the correct 3D structure because of three disulfide bonds in monomer hEGF. To address these challenges, here a fusion hEGF protein with a C terminus of collagen binding domain (CBD) along with intein protein with self-splicing property and ELP sequence was constructed by a three-step cloning procedure. This enabled us to purify recombinant hEGF without using chromatography columns. Following the confirmation of the construct by colony PCR, restriction enzymes analysis and sequencing, the 62[Formula: see text]kDa band of ELP-INTEIN-hEGF-CBD were observed on SDS-PAGE and confirmed by western blotting. Subsequently, the mitotic activity in Balb/c 3T3 cells proliferation in presence of recombinant hEGF-CBD compared with commercial hEGF using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay which showed that our purified recombinant protein stimulates the cell proliferation similar to the commercial protein. Our strategy could be considered as a new feasible approach to produce hEGF in E. coli for pharmaceutical and clinical applications.