Recombinant rat fibroblast growth factor-16: structure and biological activity.

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.

Structure and expression of a novel member, FGF-16, on the fibroblast growth factor family.

We have isolated cDNA encoding a novel member (207 amino acids) of the FGF family from the rat heart by homology-based polymerase chain reaction. As this protein is the 16th documented member of the FGF family, we tentatively term it FGF-16. Among FGF family members, FGF-16 is most similar (73% amino acid identity) to FGF-9. We have also determined the structure of human FGF-16 with high amino acid sequence identity (98.6%) to rat FGF-16. Although the predicted FGF-16 amino acid sequence lacks a typical signal sequence, recombinant rat FGF-16 was efficiently secreted by Sf9 insect cells infected with recombinant baculovirus containing the cDNA. FGF-16 mRNA was predominantly expressed in the rat heart among the adult major tissues examined. The expression profile of FGF-16 mRNA was quite different from those of other members of the FGF family. In rat embryos, FGF-16 mRNA was predominantly expressed in the brown adipose tissue. However, the expression decreased greatly after birth. These results indicate that FGF-16 in embryos might play a role in development of the brown adipose tissue.