Shahin Hadadian1,
Hasan Mirzahoseini2 
,
Dariush Norouzian Shamassebi3,
Mohamad Ali Shokrgozar4,
Saeid Bouzari5,
Saeme Asgari2
For correspondence:- Hasan Mirzahoseini Email: mirzahoseini@yahoo.com Tel:+982166480780
Received: 18 July 2014 Accepted: 8 September 2014 Published: 19 October 2014
Citation:
Hadadian S, Mirzahoseini H, Shamassebi DN, Shokrgozar MA, Bouzari S, Asgari S.
Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and ex
© 2014 The authors.
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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.
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