Production and characterization of an analog of acidic fibroblast growth factor with enhanced stability and biological activity.
We have used recombinant DNA methods to produce two forms of bovine acidic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at position 93. Both forms were expressed at high levels in Escherichia coli and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dichroic and infrared spectra suggested that the proteins are similar in secondary and tertiary structures and contain little or no alpha-helical conformations. Hydrophobic interaction chromatography showed that aFGF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, suggesting that residue 93 is exposed to the solvent. Half-maximal activity in an in vitro bioassay system was reached at a 10- to 20-fold lower dose for the aFGF C47A/H93G form than for the aFGF C47A form, suggesting that alteration of this residue has an effect on the region responsible for receptor binding. Addition of 50 micrograms/ml heparin enhanced the in vitro activity of the aFGFs, reducing the half-maximal dose to approximately 100 pg/ml for both forms, comparable to that observed previously for basic FGF with or without heparin in this assay system.
Arakawa
T
T
Amgen Inc., Amgen Center, Thousand Oaks, CA 91320-1789.
Horan
T P
TP
Narhi
L O
LO
Rees
D C
DC
Schiffer
S G
SG
Holst
P L
PL
Prestrelski
S J
SJ
Tsai
L B
LB
Fox
G M
GM
D016475:3T3 Cells / Q000187:drug effects; D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D002455:Cell Division / Q000187:drug effects; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D016220:Fibroblast Growth Factor 1 / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006358:Hot Temperature; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011489:Protein Denaturation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1; D006639:Histidine; D003545:Cysteine; D000409:Alanine; D005998:Glycine