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Original Research Article | OPEN ACCESS

Chemoselective PEGylation of Cysteine Analogs of Human Basic Fibroblast Growth Factor (hbFGF) - Design and expression

Shahin Hadadian1, Hasan Mirzahoseini2 , Dariush Norouzian Shamassebi3, Mohamad Ali Shokrgozar4, Saeid Bouzari5, Saeme Asgari2

1Quality Control Department, Research and Production Complex, Pasteur Institute of Iran, Karaj; 2Medical Biotechnology Department, Biotechnology Research Center; 3Pilot Biotechnology Department; 4National Cell Bank of Iran; 5Molecular Biology Unit, Pasteur Institute of Iran, Tehran, Iran.

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 expression. Trop J Pharm Res 2014; 13(10):1601-1607 doi: 10.4314/tjpr.v13i10.5

© 2014 The authors.
This is an Open Access article that uses a funding model which does not charge readers or their institutions for access and distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) and the Budapest Open Access Initiative (http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited..

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

Impact Factor
Thompson Reuters (ISI): 0.523 (2021)
H-5 index (Google Scholar): 39 (2021)

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