| Autor: |
Danilenko, Dimitry M., Montestruque, Silvia, Philo, John S., Li, Tiansheng, Hill, David, Speakman, Jamie, Bahru, Menbere, Zhang, Maosheng, Konishi, Morichika, Itoh, Nobuyuki, Chirica, Madaline, Delaney, John, Hernday, Natasha, Martin, Frank, Hara, Shinichi, Talvenheimo, Jane, Narhi, Linda O., Arakawa, Tsutomu |
| Zdroj: |
Archives of Biochemistry and Biophysics; January 1999, Vol. 361 Issue: 1 p34-46, 13p |
| Abstrakt: |
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 intoEscherichia colifor 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, duringE. colifermentation, 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 inE. 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 β-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 proliferationin vitro,and induces hepatocellular proliferation and increased liver weightin vivo.In summary, FGF-16, a novel FGF family member, has both unique structural and biological properties. |
| Databáze: |
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