Expression And Characterization Of Recombinant Truncated Keratinocyte Growth Factor

Keratinocyte growth factor(KGF) is a member of the fibroblast growth factor family. By binding with its receptor KGFR, KGF exerts proliferative and differentive effects on a variety of epithelial cells. It plays a key role in repairing processes of various tissues and organs. It also acts as a protective factor in injury induced by radiation and chemotherapy. The truncated KGF(KGF_des1-23) is more stable and has higher activity .In order to produce KGF_des1-23 with high purity and activity, several procaryotic strains was constructed base on different expression methods of fusion expression, secrectory expression and single protein expression. The pichia pastoris strains producing KGF_des1-23 intracellularly or extracellularly were also constructed. The details of protein expression in several strains were analyzed .The fermentation of the expression strains were observed. The purification of the inclusion body , soluble protein were explored. The proliferative effect of KGF_des1-23 on epithelial cell and mesenchymal cell were also detected.As a result , two procaryotic expression strains were gained. The fermentation of the expression strains were stable. The purity of the product got a level as high as 95% by HPLC. The protein sequence was confirmed by Edman degradation and western blotting. The data revealed that rhKGF_des1-23 exerted proliferative effect on epithelial cell.The spatial structure of KGF_des1-23 mutants was modeled by computer. The change of the Root Mean Square Deviation of atom distance caused by matution were analyzed. The important site was found out and confirmed by gene recombination technology.