The Study Of The PEGylation Of Recombinant Human Keratinocyte Growth Factor-I And The Bioactivities In Vivo And Vitro

Objective: To study the modification and purification process ofRecombinant Keratinocyte Growth Factor-I mutant deleted23amino acids onN-terminus(△N23KGF-I,△K23)，expressed in E.coli and their bioactivities in vivo andvitro. Methods:(1)Break the bacteria, purify the the protein by SP SepharoseF.F.,renaturation and Heparin Sepharose F.F.;(2) modify the protein withmPEG-maleimide(mPEG-Mal) on the free sulphydryl, and purify it,digeste it withtrypsin, and measure by LC-MS; The sites of PEGylation were confirmed by comparingthe relative peptide segments in the reduced and non-reduced peptide massmapping(PMM).(4) Evaluate the bioactivities of the protein in vivo and in vitro by NBL-7cell line and fluorouracil induced acute intestinal injuring model in rats.Results: Theprotein(△K23) purity from the processing is about95%,and the process is fixed.Research the modification process,and get two products: mPEG-Mal-△K23-40(PEG-K23-40)、mPEG-Mal-△K23-102(PEG-K23-102). In the first product,Cys40is modified bymPEG-Mal-20K;in the other,Cys102is modified.△K23about100mg is modified and thepurity also reach above95%and the modification site is single. And the activties of△K23,PEG-K23-40and PEG-K23-102keep about98%,65%and30%in contrast to KGF-1standard product for actcity measureed by NBL-7cell lines.The activties of△K23andPEG-K23-40are clear in rats mucosa injurying experiment in contrast to the controlgroup.But PEG-K23-102is not sure.Conclusion： In unconventional PEGylationcondition,fisrtly,modify the two cysteines of△K23with mPEG-Mal-20K and got twoproducts,high purity and single modification site: PEG-K23-40,PEG-K23-102. The activityof PEG-K23-40in vitro is proved preliminaryly effeciently and keep about65%activity tothe standard product.The repairing of PEG-K23-40is proved effectively in contrast to thecontrol group in intestinal mucosa injurying experiment and may be a long-acting drugcandidate. But the effect of PEG-K23-102is not clear under the same condition. Andanother action need more experiments in other condition.