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Original Research Article
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 and 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:
Email:
mirzahoseini@yahoo.com; Tel:
+98 21 66480780; Fax: +98 21 88376421
Received: 18 July 2014
Revised accepted: 8
September 2014
Tropical
Journal of Pharmaceutical Research, October 2014;
13(10): 1601-1607
http://dx.doi.org/10.4314/tjpr.v13i10.5
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 |
