title: Recombinant rat fibroblast growth factor-16: structure and biological activity.
abstract: Fibroblast growth factor-16 (FGF-16) is the most recent member of the FGF family to be cloned. Since the biologic activity of rat FGF-16 (rFGF-16) was unknown, and this protein has no apparent signal sequence, we transformed its entire cDNA into Escherichia coli for high-level expression and further characterization of this novel protein. An attempt was made to purify the expressed protein from the supernatant of mechanically lysed cells using sequential cation-exchange chromatography. This resulted in a gradual loss of the protein as precipitate throughout the purification process. In addition to precipitation during purification, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the partially purified materials showed a cluster of protein bands around 20k to 29k. Sequence analysis of the major bands indicated that two N-terminal truncations had occurred, during E. coli fermentation, purification, or both. The largest truncation resulted in the removal of the 34 N-terminal amino acids, including the initiation codon methionine. We cloned d34 rFGF-16, expressed the gene in E. coli, and developed a purification process for this form. Even with this truncated form, precipitation was a problem. We were largely able to overcome this problem, however, by including EDTA throughout the purification process. We have characterized the structure of purified d34 rFGF-16 extensively using circular dichroism, Fourier transform infrared spectroscopy, and sedimentation velocity analysis. These studies revealed that the protein has a distinct tertiary structure, consists primarily of beta-strands, has a weak tendency to self-associate, and is fairly extended. We then performed biologic assays which showed that d34 rFGF-16 induces oligodendrocyte proliferation in vitro, and induces hepatocellular proliferation and increased liver weight in vivo. In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties.
authors.affiliation: Amgen Inc., Amgen Center, Thousand Oaks, California, 91320, USA. ddanilen@amgen.com
authors.forename: D M
authors.initials: DM
authors.lastname: Danilenko
authors.forename: S
authors.initials: S
authors.lastname: Montestruque
authors.forename: J S
authors.initials: JS
authors.lastname: Philo
authors.forename: T
authors.initials: T
authors.lastname: Li
authors.forename: D
authors.initials: D
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Speakman
authors.forename: M
authors.initials: M
authors.lastname: Bahru
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.forename: M
authors.initials: M
authors.lastname: Konishi
authors.forename: N
authors.initials: N
authors.lastname: Itoh
authors.forename: M
authors.initials: M
authors.lastname: Chirica
authors.forename: J
authors.initials: J
authors.lastname: Delaney
authors.forename: N
authors.initials: N
authors.lastname: Hernday
authors.forename: F
authors.initials: F
authors.lastname: Martin
authors.forename: S
authors.initials: S
authors.lastname: Hara
authors.forename: J
authors.initials: J
authors.lastname: Talvenheimo
authors.forename: L O
authors.initials: LO
authors.lastname: Narhi
authors.forename: T
authors.initials: T
authors.lastname: Arakawa
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001681:Biological Assay; D002942:Circular Dichroism; D003433:Crosses, Genetic; D005260:Female; D005346:Fibroblast Growth Factors / Q000008:administration & dosage / Q000737:chemistry* / Q000302:isolation & purification; D006868:Hydrolysis; D007274:Injections, Intraperitoneal; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D009836:Oligodendroglia / Q000187:drug effects / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry* / Q000302:isolation & purification; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: C484686:Fgf16 protein, mouse; C110776:Fgf16 protein, rat; D011994:Recombinant Proteins; D005346:Fibroblast Growth Factors; D010447:Peptide Hydrolases