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Tropical Journal of Pharmaceutical Research
Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, Nigeria
ISSN: 1596-5996
EISSN: 1596-9827
Vol. 13, No. 10, 2014, pp. 1601-1607
Bioline Code: pr14220
Full paper language: English
Document type: Research Article
Document available free of charge

Tropical Journal of Pharmaceutical Research, Vol. 13, No. 10, 2014, pp. 1601-1607

 en Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and Expression
Hadadian, Shahin; Mirzahoseini, Hasan; Shamassebi, Dariush Norouzian; Shokrgozar, Mohamad Ali; Bouzari, Saeid & Asgari, Saeme

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 Arg81 were 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

 
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