Purification and characterization of recombinant pufferfish (Takifugu rubripes) leptin.
Synthetic cDNA encoding pufferfish (Takifugu rubripes) leptin (pfLEP) was prepared according to the published sequence. The pfLEP, transformed into Escherichia coli and expressed upon induction with nalidixic acid, was found almost entirely in the insoluble inclusion bodies (IBs). The proteins were solubilized, refolded and purified to homogeneity by anion-exchange chromatography and gel-filtration. The respective yield of dimers and monomers was 50-100mg from 5L of fermentation culture. Circular dichroism analyses revealed similarity of the purified pfLEP secondary structure to that of mammalian leptins. The purified monomers and dimers showed a single band of approximately 15 kDa following SDS-PAGE in the presence of reducing agent, whereas the dimer showed one band of approximately 30 kDa in the absence of reducing agent, indicating its formation by S-S bonds. The purified product also showed a single peak following gel-filtration under nondenaturating conditions and reverse-phase chromatography. Monomeric and dimeric pfLEPs were stable for at least 6 months in sterile solution frozen at -20 degrees C or as lyophilized powder. Both pfLEPs were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin (hLEP) receptor, but their activity was four to five orders of magnitude lower than that of hLEP. The specificity of this activity was further evidenced by its complete inhibition by hLEP antagonist. In contrast to mammalian leptins, neither form of pfLEP bound to or formed 1:1 complex with chicken leptin-binding domain, likely due to low affinity. No specific binding of either ovine or pufferfish leptins to tilapia liver membranes was detected. This work is the first report on the purification of leptin from any fish species.
Yacobovitz
M
M
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.
Solomon
G
G
Gusakovsky
E E
EE
Levavi-Sivan
B
B
Gertler
A
A
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000235:genetics; D020738:Leptin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D023721:Tetraodontiformes / Q000378:metabolism*
D020738:Leptin; D011994:Recombinant Proteins; D004247:DNA