Soluble Expression of Recombinant Human Mitsugumin 53 in Escherichia coli NiCo21(DE3).

Mitsugumin 53 (MG53) shows significant potential as a novel therapeutic agent, offering a variety of benefits, including membrane repairment, antiviral properties, and possible applications in cancer therapy. However, this protein tends to form inclusion bodies when expressed in the bacterial system, despite lacking disulfide bonds. Consequently, multiple stages—such as solubilization-tag removal, protein solubilization, purification, and refolding—are necessary to obtain an active protein, leading to significantly high production costs. In this study, we optimized both the intrinsic and extrinsic factors for the soluble expression of recombinant human MG53 (rhMG53) in Escherichia coli without a solubilization tag, subsequently assessing its wound-healing activity against HaCaT cells. The codon-optimized mg53 gene, with 5’ mRNA folding free energy (5-ΔG) higher than − 10 kcal/mol, was cloned into a T7-based expression vector and transformed into E. coli NiCo21(DE3) for protein expression. Several transformants were screened to identify high-expressing colonies. To optimize the expression of rhMG53, we varied the incubation temperature, inducer concentration, and pre-induction growth conditions. The SDS-PAGE results demonstrated that the soluble 53 kDa MG53 protein was successfully expressed under optimal conditions at 25 °C, using 0.5 mM IPTG and induced at an optical density of 0.8. An in vitro scratch assay revealed that the scratch wound was almost completely closed within 24 h with an optimum concentration of 60 µg/mL rhMG53. Techno-Economic Analysis showed that our approach can drastically lower production costs, reducing them by one-fourth compared to the inclusion body approach. The findings highlight the successful soluble expression of MG53 in a bacterial system without the use of a solubilization tag.