Characterization of a biologically active extracellular domain of fibroblast growth factor receptor 1 expressed in Escherichia coli.
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.
Bergonzoni
L
L
Department of Biotechnology, Farmitalia Carlo Erba, Milan, Italy.
Caccia
P
P
Cletini
O
O
Sarmientos
P
P
Isacchi
A
A
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1; D011956:Receptors, Cell Surface / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011956:Receptors, Cell Surface; D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D016222:Fibroblast Growth Factor 2; C496348:FGFR1 protein, human; D011505:Protein-Tyrosine Kinases; D020794:Receptor Protein-Tyrosine Kinases; D051496:Receptor, Fibroblast Growth Factor, Type 1