Expression、purification And Bioactivity Of Recombinant Human Keratinocyte Growth Factor-1

Keratinocyte growth factor 1(KGF-1) which is also known as fibroblast growth factor (FGF7) is a member of fibroblast growth factor family(FGFs).Its specific receptor FGFR2Ⅲb is only expressed in the epithelial cells, so KGF-1 is acting on the epithelial cells specifically.This characteristic makes the drug containing KGF-1 have better directionality and security.In 2004, the drug of AMGEN company-Palifermin injection (Kepivance) - was approved by FDA.The injection is mainly used in treating oral mucositis caused by radiotherapy and chemotherpy.Fuethermore there are still some potential applications for chronic wound healing caused by metabolic diseases,a major obstacle in preterm infants who lacking of surfactant and bronchopulmonary dysplasia and the treatment of acute lung injury and acute liver injury.Although KGF-1 has great potentials for application in medicine,the research of KGF-1 expression is still in the exploration stage.KGF-1 was attempted to express in E.coli,CHO cells,plant cells and others expressiong system,but the results were not satisfactory.SUMO has similar structures with the ubiquitin protein. Unlike the ubiquitination,the sumoylation in vivo can enhance the stability of the target proteins,help the target proteins to transfer and fold correctly, and also have a effect on their activities.In these years, the SUMO tag has been widely used to express the proteins which was difficult to express directly, significantly increased expression quantity and the soluble form of these proteins.Therefore,the feasibility of using SUMO tag to express KGF-1 in fusion protein form was studied. Firstly, the sequence encoding △N23 rhKGF-1 (the form of KGF-1 missing 23amino acids in N terminal) was synthesized and fused with SUMO through overlap PCR to obtain 6His-sumo-rhKGF-1 fusion gene.Then the sequences of rhKGF-1 and 6His- -sumo-rhKGF-1 were subclone into expression vectors pJW2 and pET30a. The ecombinant expression vectors were transformed into E.coli BL21(DE3) to produce the recombinant protein and screen out the most efficient recombinant expression plasmid.Then,this plasmid was codon optimized， while the inducing conditions were changed to improve the quantity of the recombinant protein expression. After inducing expression， the engineering bacterium was crushed through ultrasonic,took the supernatant purified by Ni-NTA.Collected the purified fusion protein,then the sumo tag was digested using SUMO Protease.After that,the mixed liquor was purified by Ni-NTA again,to obtain purified rhKGF-.The biological activity of rhKGF-1 was detected using HaCat cell model.Four results were obtaned in the following:Firstly,restriction analysis and sequencing proved recombinant expression plasmids pET30a-rhKGF-1,pET30a-6His-sumo-rhKGF-1,pJW2-rhKGF-1,pJW2-6His-sumo-rhKGF-1 were constructed correctly.The positive recombinant expression plasmid is pET30a-6His-sumo-rhKGF-1.Secondly,neither the codon optimization nor the changing of induce conditions could improve the quantity of rhKGF-1 expression. Thirdly,the results of SDS-PAGE displayed that the recombinant proteins mostly existed in form of soluble protein.Eight milligram fusion protein were obtained from four litres of fermented liquid.Finally,after purification and enzyme digestion to the fusion protein,rhKGF-1, which has the activity to promote HaCat cell proliferation,was obtained. EC50= 6.11ng/mL.In this study,The soluble form of rhKGF-1,which is expressed in E.coli,was increased.The bioactive AN23 KGF-1 was obtaine,that was to provide experimental basis for its further pharmaceutical, preparation and data support.