Expression Of Keratinocyte Growth Factor In Escherichia.coli And Its Purification And Bioactivity

Keratinocyte Growth Factor (KGF) is a member of Fibroblast Growth Factor(FGF-7). By acting predominantly in a paracrine manner, KGF can stimulate the proliferation, differentiation and migration of epithelial cells. A serious studies have provided insight into the function of KGF in repair processes of various tissues and organs, including skin, cornea, bowel. In addition, it is a protective factor for gastrointestinal injury induced by radiation and chemotherapy. Interest in producing large amount and bioactive KGF through genetic engineering method has been heightened due to the limited amount of KGF in the body. Based on previous work, this paper focus on the expression, purification and bioactive test of KGF for the sake of providing groundwork for future study.By changing the inducing temperature, inducer IPTG concentration, the density of bacteria and inducing time, the satisfying condition for expression of soluble fusion protein GST-KGF could be obtained at 25 0C, 0.3mmolIL IPTG, QD~ equals to 0.97, 5h inducing time. Under the improved condition, the soluble GST-KGF comprised about 4.2% of total cellular protein and 25mg GST-KGF could be harvested in IL fermentation.A method which combined two-step of affinity chromatography and trypsin incubation was used to achieve KGF purification since the fusion protein GST-KGF could bind to GSH-Sepharose 4B and Heparin-Sepharose CL-6B and Heparin-Sepharose CL-6B protects the active core of KGF from trypsin proteolysis. the 1 7KD KGF derived from GST-KGF was purified to homogeneity. 5.2mg purified KGF, a total recovery of 52% KGF, was harvested. Western-Blot analysis showed that the purified KGF could be detected by rabbit against human KGF.MTT assay was used to investigate the effect of recombined KGF on the proliferation of mouse comeal epithelial cells, which were cultured throughABSTRACT-111-tissue culture method. Results showed that KGF enhanced cellular proliferation in a dose dependent manner: when the concentration of KGF was 0.1 ng/mL, it began to show the ability to stimulate the proliferation of culture epithelial cells(P<0.05), when the concentration increased to lOng/niL, it showed the most intensive proliferative effect on cultured cells. However, this kind of effect decreased when the concentration of KGF increased to 1 OOngImL.The effects of KCIF were evaluated on the model of scalded wounds n mice. Results showed that the topical application of KGF could accelerate wound healing: the wound area of treated group was smaller than the control one at the same time after scalded; the average healing time was (24.5±1.7) days for treated groups while (21.2±1.2) days for control one.