title: A novel solid-phase site-specific PEGylation enhances the in vitro and in vivo biostabilty of recombinant human keratinocyte growth factor 1.
abstract: Keratinocyte growth factor 1 (KGF-1) has proven useful in the treatment of pathologies associated with dermal adnexae, liver, lung, and the gastrointestinal tract diseases. However, poor stability and short plasma half-life of the protein have restricted its therapeutic applications. While it is possible to improve the stability and extend the circulating half-life of recombinant human KGF-1 (rhKGF-1) using solution-phase PEGylation, such preparations have heterogeneous structures and often low specific activities due to multiple and/or uncontrolled PEGylation. In the present study, a novel solid-phase PEGylation strategy was employed to produce homogenous mono-PEGylated rhKGF-1. RhKGF-1 protein was immobilized on a Heparin-Sepharose column and then a site-selective PEGylation reaction was carried out by a reductive alkylation at the N-terminal amino acid of the protein. The mono-PEGylated rhKGF-1, which accounted for over 40% of the total rhKGF-1 used in the PEGylation reaction, was purified to homogeneity by SP Sepharose ion-exchange chromatography. Our biophysical and biochemical studies demonstrated that the solid-phase PEGylation significantly enhanced the in vitro and in vivo biostability without affecting the over all structure of the protein. Furthermore, pharmacokinetic analysis showed that modified rhKGF-1 had considerably longer plasma half-life than its intact counterpart. Our cell-based analysis showed that, similar to rhKGF-1, PEGylated rhKGF-1 induced proliferation in NIH 3T3 cells through the activation of MAPK/Erk pathway. Notably, PEGylated rhKGF-1 exhibited a greater hepatoprotection against CCl(4)-induced injury in rats compared to rhKGF-1.
authors.affiliation: Key Laboratory of Biotechnology and Pharmaceutical Engineering of Zhejiang Province, Wenzhou Medical College, Wenzhou, China.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Huang
authors.forename: Guanghui
authors.initials: G
authors.lastname: Zhu
authors.forename: Chuanchuan
authors.initials: C
authors.lastname: Sun
authors.forename: Jingui
authors.initials: J
authors.lastname: Zhang
authors.forename: Yi
authors.initials: Y
authors.lastname: Zhang
authors.forename: Youting
authors.initials: Y
authors.lastname: Zhang
authors.forename: Chaohui
authors.initials: C
authors.lastname: Ye
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Wang
authors.forename: Dariush
authors.initials: D
authors.lastname: Ilghari
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Li
mesh_terms: D000447:Aldehydes / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002251:Carbon Tetrachloride / Q000009:adverse effects; D056486:Chemical and Drug Induced Liver Injury / Q000517:prevention & control; D019610:Cytoprotection / Q000187:drug effects; D051523:Fibroblast Growth Factor 7 / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D006801:Humans; D017114:Liver Failure, Acute / Q000139:chemically induced / Q000517:prevention & control; D008297:Male; D010446:Peptide Fragments / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D055550:Protein Stability; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology*; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000447:Aldehydes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D051523:Fibroblast Growth Factor 7; D011092:Polyethylene Glycols; D002251:Carbon Tetrachloride; C018475:butyraldehyde