Refolding of bioactive human epidermal growth factor from E. coli BL21(DE3) inclusion bodies & evaluations on its in vitro & in vivo bioactivity

Abstract

Human epidermal growth factor (hEGF) is a mitogenic protein widely used in pharmaceutical and cosmetic industries, thus recombinant DNA technology has been applied to meet the high demand for hEGF. The overexpression of recombinant protein in E. coli often leads to the formation of inclusion bodies (IBs). Mild solubilisation preserves the native secondary protein structure in IBs, thereby the high recovery of active protein from IBs. The redox system also plays a pivotal role in the formation of disulphide bonds during refolding of disulphide bond-containing protein. This study aimed to recover hEGF from bacterial IBs through freeze-thawing solubilisation and glutathione-based oxidative refolding. CBD-Ssp DnaB-hEGF fusion protein was expressed as IBs in E. coli, washed with Triton X-100 and urea to remove most protein contaminants, then the solubilised fusion protein was obtained by freeze-thawing with the addition of 2 M urea. The solubilised protein was subsequently refolded by intein cleavage via a glutathione-based redox system. The refolded hEGF demonstrated heat-resistant properties, interacted with specific antibodies on ELISA, stimulated keratinocyte proliferation and possessed significant in vivo wound healing properties on the 8th day, confirming that hEGF was correctly folded. In summary, the protocol described is suitable for the recovery of refolded hEGF from bacterial IBs by mild solubilisation and oxidative refolding.

Keywords

• E. coli BL21(DE3)
• Heat-resistant
• hEGF
• Freeze-thawing
• Glutathione-based redox system