Efficient recovery of bioactive recombinant human epidermal growth factor from inclusion bodies via Temperature-assisted Denaturation.

Human epidermal growth factor (hEGF) is a potent mitogen used for wound healing and skin rejuvenation. To meet the rising demand for hEGF, recombinant hEGF (rhEGF) is commonly produced by expression in Escherichia coli; however, the overexpression results in the formation of inclusion bodies (IBs). Subsequently, the conventional method to recover bioactive rhEGF from IBs requires high concentrations of denaturant, which are resource-intensive and harmful to the environment. In this study, we developed a Temperature-assisted Denaturation (TAD) method that uses thermal energy to disrupt the hydrophobic interactions between misfolded proteins in IBs to maximize the recovery of bioactive rhEGF with minimum denaturant. A sequence of Design of Experiment (DoE) approach using factorial design and response surface methodology was performed to optimize the TAD method. Using TAD, we achieved a three-fold yield improvement over the chemical denaturation methods. The TAD method also demonstrated better process efficiency by reducing buffer consumption and processing time. Following a two-stage purification process (anion-exchange chromatography and ultrafiltration), we achieved a 71.5% purification yield of nucleic acid-free, bioactive rhEGF with a final purity of ≥ 93%. These results show that thermal assistance with minimum denaturant use during the solubilization stage is a viable method for the recovery of bioactive rhEGF from its IBs.