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. 2015 Aug 31:4:176.
doi: 10.4103/2277-9175.164001. eCollection 2015.

Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

Shahin Hadadian  1 , Dariush Norouzian Shamassebi  2 , Hasan Mirzahoseini  3 , Mohamad Ali Shokrgozar  4 , Saeid Bouzari  5 , Mina Sepahi  6
Affiliations

Stability and biological activity evaluations of PEGylated human basic fibroblast growth factor

Shahin Hadadian et al. Adv Biomed Res. .

Abstract

Background: Human basic fibroblast growth factor (hBFGF) is a heparin-binding growth factor and stimulates the proliferation of a wide variety of cells and tissues causing survival properties and its stability and biological activity improvements have received much attention.

Materials and methods: In the present work, hBFGF produced by engineered Escherichia coli and purified by cation exchange and heparin affinity chromatography, was PEGylated under appropriate condition employing 10 kD polyethylene glycol. The PEGylated form was separated by size exclusion chromatography. Structural, biological activity, and stability evaluations were performed using Fourier transform infrared (FITR) spectroscopy, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay and effect denaturing agent, respectively.

Results: FITR spectroscopy revealed that both PEGylated and native forms had the same structures. MTT assay showed that PEGyalated form had a 30% reduced biological activity. Fluorescence spectrophotometry indicated that the PEGylated form denatured at higher concentrations of guanidine HCl (1.2 M) compared with native, which denatured at 0.8 M guanidine HCl.

Conclusions: PEGylation of hBFGF makes it more stable against denaturing agent but reduces its bioactivity up to 30%.

Keywords: 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay; Fourier transform infrared spectroscopy; human basic fibroblast growth factor; polyethylene glycol methyl ether maleimide; stability.

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Conflict of interest statement

Conflict of Interest: None declared.

Figures

Figure 1
Figure 1
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results of purified human basic fibroblast growth factor (hBFGF). Lane A: Ladder, Lane B: Heparin affinity eluate, Lane C: Cation exchange eluate, Lane D: Western of standard hBFGF, Lane E: Western of purified hBFGF
Figure 2
Figure 2
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results of two fraction separated by size exclusion chromatography. Lane A: PEGylated form in first fraction, Lane B: Non-PEGyalated form in second fraction, Lane C: Ladder
Figure 3
Figure 3
Biological activity analysis. Left: Proliferation assay for standard human basic fibroblast growth factor (hBFGF) (-♦-), PEGylated hBFGF (▪▪▫▪) and non-PEGylated form (-▴-). Right: Comparison of proliferation rate of PEGylated non-PEGylated hBFGF
Figure 4
Figure 4
Fluorescence spectrophotometry. (a) intact PEGylated basic fibroblast growth factor (BFGF), (b) denatured form of PEGylated BFGF
Figure 5
Figure 5
Fourier transform infrared (FITR) spectroscopy of free basic fibroblast growth factor-polyethylene glycol (BFGF-PEG), BFGF and PEG. (a) FTIR of PEGylated form, (b) FTIR of standard BFGF and PEG 10 kDa
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