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Published:
Dec 9, 2014Keywords:
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Main Article Content
Chemoselective PEGylation of cysteine analogs of human basic fibroblast growth factor (hbFGF) - design and expression
Abstract
Purpose: To improve the stability and bioactivity of human basic fibroblast growth factor (hbFGF) by site-specific pegylation
Methods: Four new mutants of hbFGF were designed with substituted Asp68, Lys77, Glu78 and Arg81 with cysteine with the aid of bioinformatics technique, and then cloned into pET21a plasmid, transferred into E. coli BL21 (DE3). The expressed proteins were purified using cation exchange and heparin affinity chromatography. Cysteine analogs of hbFGF were PEGylated with 10 KDa PEG and purified using size exclusion chromatography. Mitogenic activity and resistance against denaturation agents were evaluated by MTT assay and fluorescence spectrophotometry, respectively, and the results obtained were compared with the non-PEGylated form.
Results: Despite greater resistance against denaturation agent (1.2 M guanidine hydrochloride for denaturation of PEGylated mutants compared with 0.8 M for non-PEGylated forms), the mitogenic activities of the four mutants Asp68, Lys77, Glu78 and Arg81were retained at 79, 78.6, 83.3 and 75.6 %, respectively.
Conclusion: PEGylated hbFGF shows decreased mitogenic activity and increased resistance against denaturation agent.
Keywords: Bioinformatics, Fibroblast growth factor, Cysteine analog, PEGylation, Denaturation agent, Guanidine hydrochloride, Mitogenic activity
