Stabilization of recombinant human basic fibroblast growth factor by chemical modifications of cysteine residues.
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.
Caccia
P
P
Department of Biotechnology, Farmitalia Carlo Erba, Milan, Italy.
Nitti
G
G
Cletini
O
O
Pucci
P
P
Ruoppolo
M
M
Bertolero
F
F
Valsasina
B
B
Roletto
F
F
Cristiani
C
C
Cauet
G
G
D000818:Animals; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000235:genetics / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013438:Sulfhydryl Compounds / Q000378:metabolism
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D016222:Fibroblast Growth Factor 2; D006493:Heparin; D004247:DNA; D003545:Cysteine