Co-expression of Recombinant Human Epidermal Growth Factor (rhEGF) In Escherichia coli BL21 (DE3) with Bacillus cereus Phospholipase C

摘要

Diabetes mellitus is a metabolic disorder in the body caused by high levels of blood sugar. Diabetic foot ulcer is a further complication due to ulceration in the feet of diabetic patients where more than 10% of patients must be amputated. To prevent this, a therapeutic agent is needed in order to treat and prevent ulceration. Human Epidermal Growth Factor is a protein which is known having biological functions to accelerate cell proliferation and can be used as a candidate for wound healing agents. This paper studies the production of recombinant hEGF by co-expression with Bacillus cereus phospholipase C (PLC). The research method began with the construction of plasmid pETDuet-1-PLC-hEGF, host cell transformation and optimization of Isopropyl-β-D-thiogalactopiranoside (IPTG) concentration as the inducer. Co-expression results were characterized using Tricine SDS-PAGE and western blot, while hEGF protein levels were determined using enzyme linked immunosorbent assay (ELISA). The results showed that recombinant hEGF and PLC enzyme were secreted to the growth medium in 60 hours after induction with the optimum IPTG concentration 0.1 mM. The results of characterization with Tricine SDS-PAGE showed the presence of PLC (28 kDa) and hEGF (6.2 kDa) bands which were confirmed by western blot using anti-mouseEGF. 503.48 μg/mL of hEGF concentration was successfully measured in the culture medium using ELISA and it was slightly higher than other previous studies.