1. Utilization of Freeze-Thawing Method for High-level Expression of Functional Human Epidermal Growth Factor (hEGF)
- Author
-
Mohammadi, Mozafar, Ghanbari, Saeid, Emamgholi, Asgar, and Hashemzadeh, Mohammad Sadegh
- Abstract
The wide applications of human epidermal growth factor (hEGF) as an efficient mitogen agent in the clinical, health, and cosmetic industries has made it an interesting research target. But its low abundance in natural resources has limited its utilization. Therefore, the high-level expression of recombinant hEGF (rhEGF) has received attention in recent years. However, expression of rhEGF in the Escherichia coli intracellular environment often results in the formation of inclusion bodies (IBs). In this study, to increase the expression level and solubility, the SUMO tag was fused to the mature hEGF gene and expressed in E. coli BL21 (DE3). Subsequently, the freeze-thawing method plus 2 M urea solubilized IBs. Further, the SUMO-rhEGF protein was purified by Ni-NTA affinity chromatography, and the biological activity of the purified protein was investigated on the NIH3T3 cell line by MTT assay. The results showed SUMO tag succeeded in increasing the fusion protein expression level, but ~80% of them were in the form of IBs. Further, it was found the freeze-thawing method could significantly solubilize and refold about 70% of the IBs. MTT assays also indicated the different concentrations of rhEGF had mitogenic activity in a dose-dependent manner and significantly increases the viability percentage of treated cells after 48 h. The results of this study showed that the freeze-thawing method has improved the expression yield of rhEGF, and due to its cost-effectiveness and high efficiency, it can be a promising strategy to produce rhEGF for pharmaceutical and industrial development. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF