Previously, we constructed the recombinant plasmid which containing human epidermal growth factor and collagen binding domain for overproduction of fused biofunctional protein. However, this fusion protein was expressed in Escherichia coli as insoluble protein form in cytoplasm. Therefore, effective denaturation and dialysis process is critical for solubilization and refolding in protein purification process. We attempted several chemicals and buffer conditions for induction, dialysis, and solubilization. Using lactose instead of isopropyl β-D-1-thiogalactopyranoside, expression of target protein was induced. 20 mM tris-HCl buffer for dialysis was suitable for the activity and soluble form of fusion protein. Furthermore, for the solubility of expressed inclusion protein, we conducted with various pH conditions and concentrations of urea and guanidine hydrochloride. The efficient solubility of inclusion body form of fusion protein was showed at alkaline pH condition containing urea.