The purpose of this study was to provide an economical and easy preparation method for recombinant human epidermal growth factor (hEGF) without the need for an expensive enzyme to cleave the fusion part. However, the N-terminal fusion part is still useful for affinity chromatography. The hEGF is an important hormone in cell growth and proliferation in humans, and many studies on the expression and purification of this protein have been reported. In the present study, the hEGF gene was designed to be optimized with the E. coli codon usage preference and to contain Asn-Gly at the N-terminus of the protein. The gene was inserted into pRSET_A, an E. coli expression vector, and transformed into E. coli BL21 (DE3). The recombinant fusion protein was successfully co-expressed with pG-Tf2, a chaperone vector, in E. coli and purified by Ni-NTA column chromatography. The rhEGF was then released by hydroxylamine treatment and confirmed by SDS-PAGE. ELISA and cell proliferation assay were measured to confirm the biological activity of recombinant hEGF, and the following conclusions were drawn.

1. ELISA analysis was performed for comparative analysis of the activity of recombinant hEGF. Standard hEGF, inclusion body of recombinant hEGF and refolding protein of recombinant hEGF were used as controls. As a result of the analysis, the recombinant hEGF co-expressed with the chaperone showed about 8% higher activity than the recombinant hEGF subjected to the refolding process.

2. Cell proliferation of recombinant hEGF was confirmed through CCD-986sk, a human fibroblast. It was confirmed that recombinant hEGF caused cell proliferation by using CCD-986sk having EGFR, and it was further confirmed that cell proliferation was caused by concentration-dependently within 10 ppm.