title: Characterization of a cysteine-free analog of recombinant human basic fibroblast growth factor.
abstract: Using oligo site-directed mutagenesis, we have modified our synthetic gene for human basic fibroblast growth factor (bFGF) to replace all four cysteine codons with serine codons. The corresponding protein was expressed in Escherichia coli and purified from inclusion bodies by solubilization in urea followed by a series of column chromatographies and a folding step. The resulting protein, having no cysteine residues, is unable to form either intramolecular or intermolecular disulfide bonds. The secondary and tertiary structures of the purified analog, as determined by circular dichroism and fluorescence spectroscopy, were identical within experimental error to recombinant bovine and human bFGF with unaltered amino acid sequences. Reflecting the similar conformation, the analog protein exhibited mitogenic activity on NIH 3T3 cells which was indistinguishable from the natural sequence molecule.
authors.affiliation: Amgen Inc., Thousand Oaks, CA 91320.
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
authors.forename: Y R
authors.initials: YR
authors.lastname: Hsu
authors.forename: S G
authors.initials: SG
authors.lastname: Schiffer
authors.forename: L B
authors.initials: LB
authors.lastname: Tsai
authors.forename: C
authors.initials: C
authors.lastname: Curless
authors.forename: G M
authors.initials: GM
authors.lastname: Fox
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005346:Fibroblast Growth Factors / Q000031:analogs & derivatives / Q000235:genetics* / Q000502:physiology; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D051379:Mice; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins / Q000031:analogs & derivatives; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013936:Thymidine / Q000378:metabolism
chemical_list: D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012694:Serine; D005346:Fibroblast Growth Factors; D003545:Cysteine; D013936:Thymidine