Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: A bona fide tracer

doi:10.1016/j.ab.2010.12.029

Analytical Biochemistry

Volume 411, Issue 1, 1 April 2011, Pages 1-9

https://doi.org/10.1016/j.ab.2010.12.029 Get rights and content

Abstract

Here we describe, for the first time, the design and characterization of a bona fide fluorescently labeled mutant of the human acidic fibroblast growth factor (aFGF). The aFGF–Cys2 mutant was recombinantly synthesized by substituting the second amino acid with a reactive cysteine whose sulfhydryl group’s side chain reactivity facilitated the covalent binding of a fluorescent probe as a thiolyte monobromobimane. Using a combination of biophysical and functional assays, we found that the fluorescently labeled mutant aFGF is characterized by essentially the same global folding, mitogenic activity, and association behavior with heparin, its physiological activator, as the unlabeled wild-type protein. We used this new tracer protein mutant to determine the association behavior of aFGF with heparin in the presence of high concentrations of albumin that mimicked more closely the plasma medium in which aFGF is naturally located and in which it has evolved to function. By exposing the aFGF–Cys2–heparin complex to increasing concentrations of albumin up to physiological plasma levels, we were able to demonstrate that macromolecular crowding does not affect the stoichiometry of the interaction. In summary, the dimeric aFGF–Cys2–heparin complex might represent a biologically relevant complex in physiological media.

Section snippets

Reagents

Restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Bacteriological agar, bactotryptone, yeast extract, Ham’s F-12, Dulbecco’s modified Eagle’s medium (DMEM), and calf serum were manufactured by Gibco (Invitrogen, Carlsbad, CA, USA). Sodium heparin (average molecular mass = 3 kDa) and bovine serum albumin (BSA) were obtained from Sigma–Aldrich (St. Louis, MO, USA). Heparin–Sepharose and Sephadex G-25 were obtained from Pharmacia Fine Chemicals (New Market, NJ, USA).

Expression and purification of aFGF–Cys2

aFGF–Cys2 was expressed from cultured cells of E. coli BL21(DE3) transformed with aFGF–Cys2–pRAT-4 plasmid and purified by affinity chromatography on a heparin–Sepharose column (see Materials and methods for details). The protein was eluted from the heparin column as a single peak with 1.5 M NaCl in 10 mM sodium phosphate (pH 7.2), similar to the wild-type form of aFGF (not shown), and was homogeneous as determined by SDS–PAGE (Fig. 1). A regular yield of 6–7 mg of pure protein was obtained per

Concluding remarks

This article has described a new procedure to generate a bona fide tracer for aFGF, known as aFGF–Cys2–mBBr, that retains the biological activity and association behavior of the wild-type form of the protein. Substitution of the second amino acid residue with a reactive cysteine residue in the N-terminal domain of aFGF that lacks a defined three-dimensional structure appeared not to affect protein folding of the new mutant given that the biological activity of the resultant protein remained

Acknowledgments

We thank Allen P. Minton (National Institutes of Health) for his help with the data analysis and for helpful discussions, and we thank Carlos Alfonso (Centro de Investigaciones Biológicas) for his assistance with the analytical ultracentrifugation experiments. This work was funded in part by Grants MAT2008-06719-C03-02 (R.M.L.), BIO2008-04478-C03 (G.R.), BFU2008-02595 (G.G.-G.), and CONSOLIDER CSD2009-00088 from the Ministry of Science and Innovation (Spain). M.J. Feito was supported by a

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Site-specific labeling and functional efficiencies of human fibroblast growth Factor-1 with a range of fluorescent Dyes in the flexible N-Terminal region and a rigid β-turn region
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Both these residues are exposed to the solvent, which should allow accessibility to the probe, but not in the region where the receptor and heparin binds, which are both required for bioactivity (Fig. 1B) [61]. A previous report showed that residue 2 could be efficiently labeled with the blue-emitting alkyl halide-functionalized dye, monobromobimane (mBBr) but not with 5-bromomethyl fluorescein (BrF) [57]. A tentative explanation for this difference was postulated based on the size of the dye, but this was not fully investigated.
Human fibroblast growth factor-1 (hFGF1) binding to its receptor and heparin play critical roles in cell proliferation, angiogenesis and wound healing but is also implicated in cancer. Fluorescence imaging is a powerful approach to study such protein interactions, but it is not always obvious if the site chosen will be efficiently labeled, often relying on trial-and-error. To provide a more systematic approach towards an efficient site-specific labeling strategy, we labeled two structurally distinct regions of the protein – the flexible N-terminus and a rigid loop. Several dyes were chosen to cover the visible region and to investigate how the structure of the dye affects the labeling efficiency. Flexibility in either the protein labeling site or the dye structure was found to result in high labeling efficiency, but flexibility in both resulted in a significant decrease in labeling efficiency. Conversely, too much rigidity in both can result in dye-protein interactions that can aggregate the protein. Importantly, site-specifically labeling hFGF1 in these regions maintained biological activity. These results could be applicable to other proteins by considering the flexibility of both the protein labeling site and the dye structure.
Expression of biologically recombinant human acidic fibroblast growth factor in Arabidopsis thaliana seeds via oleosin fusion technology
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Citation Excerpt :
Human acid fibroblast growth factor (aFGF), also called FGF-1, the physiological form of which contains 154 amino acids (Zazo et al., 1992) is a heparin binding protein. It was involved in a variety of biological processes, including angiogenesis, cell proliferation, and differentiation (Kenneth and Guillermo, 1986; Jose Feito et al., 2011). The aFGF protein is an important member of the growth factor families.
The potential of oleosins to act as carriers for recombinant foreign proteins in plant cells has been established. Using the oleosin fusion technology, the protein can be targeted to oil bodies in oilseeds by fusing it to the N- or C-terminus of oleosin. In this study, aFGF was expressed in Arabidopsis thaliana seeds via oleosin fusion technology. A plant-preferred aFGF gene was synthesized by optimizing codon usage and was fused to the C-terminus of the A. thaliana 18.5 kDa oleosin gene. The fusion gene was driven by the phaseolin promoter to confer seed-specific expression of the human acidic fibroblast growth factor in A. thaliana. The T-DNA region of the recombinant plasmid pKO-aFGF was introduced into the genome of Arabidopsis thaliana by the floral dip method. The aFGF protein expression was confirmed by SDS-PAGE and western blotting. The biological activity showed that oil bodies fused to aFGF stimulated NIH/3T3 cell proliferation activity.

¹: This article is dedicated to the memory of Concha Fernández-Cabrera, who passed away 24 February 2010.

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Strategy for fluorescent labeling of human acidic fibroblast growth factor without impairment of mitogenic activity: A bona fide tracer

Abstract

Section snippets

Reagents

Expression and purification of aFGF–Cys2

Concluding remarks

Acknowledgments

Gene

Biochem. Biophys. Res. Commun.

Cell

Methods

Arch. Biochem. Biophys.

Trends Biochem. Sci.

J. Biol. Chem.

Gene

J. Biol. Chem.

Biophys. Chem.

J. Mol. Biol.

Anal. Biochem.

Methods Enzymol.

J. Biol. Chem.

Fibroblast growth factors: broad spectrum mitogens with potent angiogenic activity

Trends Biochem. Sci.

Structure of a heparin-linked biologically active dimer of fibroblast growth factor

Nature