ABSTRACT
The extracellular domain of human fibroblast growth factor receptor (XC-FGF-R) was expressed in Escherichia coli. The protein was purified to homogeneity and the interaction with basic fibroblast growth factor (bFGF), its physiological ligand, was examined. Using resins on which bFGF was reversibly bound, we analysed the characteristics of the binding between XC-FGF-R and immobilized bFGF. We also investigated the stoichiometry of the binding between XC-FGF-R and recombinant human bFGF (rhbFGF) applying non-denaturing gel electrophoresis, chemical cross-linking followed by SDS/PAGE, and gel-filtration chromatography. In cross-linking and gel-filtration chromatography experiments, a 1:1 complex between rhbFGF and XC-FGF-R was observed. The complex was separated from the non-complexed proteins using non-denaturing PAGE in the presence of 0.1% Triton X-100. The band corresponding to the complex was recognized by specific antibodies directed against bFGF and its receptor, blotted on poly(vinylidene difluoride) membranes and submitted to sequence and amino acid analysis. The data obtained from these determinations confirmed the formation of a 1:1 complex between rhbFGF and XC-FGF-R.
Subject(s)
Fibroblast Growth Factor 2/metabolism , Receptors, Fibroblast Growth Factor/metabolism , In Vitro Techniques , Protein Binding , Recombinant Proteins , SolubilityABSTRACT
The functional features of a recombinant fibroblast growth factor (FGF) receptor (FGF-R) were investigated by expressing at high level in Escherichia coli a soluble non-glycosylated form of FGF-R1. The extracellular domain of the mature protein (XC-FGF-R), comprising the first 356 amino acids, was purified from a large-scale fermentation. After cell lysis, the protein was quantitatively found in the pellet. XC-FGF-R was solubilized using guanidine/HCl and allowed to refold using two dialysis steps. The refolded protein was obtained in a homogeneous form after ammonium sulphate precipitation and gel-filtration chromatography. The soluble receptor had the ability to form a complex with recombinant human basic FGF (rhbFGF) in solution, as demonstrated by immunoprecipitation with anti-(FGF-R) serum. Formation of a rhbFGF/XC-FGF-R complex was visualized by cross-linking experiments. Quantitative binding experiments with the XC-FGF-R immobilized on Affi-Gel resin showed high binding affinity for 125I-bFGF (Kd = 5-10 nM). Purified XC-FGF-R inhibited binding of 125I-bFGF to its high-affinity receptors on baby hamster kidney cells. These data suggest that glycosylation of the FGF-R is not necessary for its ligand-binding activity. The use of an E. coli expression system resulted in the efficient production of a soluble receptor in a form suitable for ligand/receptor structural studies and screening of new potential agonists and antagonists of angiogenesis. These results indicate that E. coli can be used for the production of complex molecules such as Ig-like receptors.
Subject(s)
Escherichia coli/genetics , Fibroblast Growth Factor 2/metabolism , Receptor Protein-Tyrosine Kinases , Receptors, Cell Surface/metabolism , Receptors, Fibroblast Growth Factor , Animals , Base Sequence , Binding, Competitive , Cell Line , Cloning, Molecular , Glycosylation , Humans , Kinetics , Molecular Sequence Data , Molecular Weight , Oligodeoxyribonucleotides , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Cell Surface/genetics , Receptors, Cell Surface/isolation & purification , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
The production of recombinant human basic fibroblast growth factor (rhbFGF) in Escherichia coli cells yielded active forms of this polypeptide which, however, displayed a high degree of instability towards oxidative processes. Biochemical studies in our laboratory and those of others indicated that the reactivity of the four cysteine residues was the main cause of the observed instability. Several attempts to obtain more stable derivatives of rhbFGF were carried out by modification of the sulfhydryl groups. Among these, treatment of rhbFGF with iodoacetic acid led to the isolation of a partially carboxymethylated form (Cm-FGF). Peptide mapping analysis of the modified protein showed that two cysteines (78 and 96) were blocked by a carboxymethyl group. The remaining cysteines (34 and 101) were not modified under the conditions used and were found to be in the reduced form. Cm-FGF and unmodified rhbFGF showed similar affinity both for heparin and for high-affinity receptors. Cm-FGF was more stable than the unmodified molecule as measured by HPLC and SDS/PAGE analysis. Interestingly, Cm-FGF was more active than unmodified rhbFGF in stimulating proliferation of endothelial cells and DNA synthesis in 3T3 fibroblasts. This new derivative could represent a desirable complementation to rhbFGF for the development of more stable pharmaceutical formulations in wound healing applications.
