Purification and characterization of recombinant pufferfish (Takifugu rubripes) leptin

General and Comparative Endocrinology
M YacobovitzArieh Gertler

Abstract

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 w...Continue Reading

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Citations

Jul 7, 2011·Indian Journal of Clinical Biochemistry : IJCB·Jian Feng LiShuang Quan Zhang
Mar 24, 2012·Animal : an International Journal of Animal Bioscience·A R G Wylie
Apr 17, 2010·General and Comparative Endocrinology·Lingjiang HeLihong Yuan
Oct 28, 2009·General and Comparative Endocrinology·Guan-Gui LiKaaseng Lai
Aug 19, 2009·General and Comparative Endocrinology·B Levavi-SivanJ J Lareyre
May 16, 2009·General and Comparative Endocrinology·Geir Lasse TarangerTom Hansen
Jun 10, 2008·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Koji MurashitaTadahide Kurokawa
May 22, 2009·Annals of the New York Academy of Sciences·M CarrilloA Gómez
May 24, 2014·General and Comparative Endocrinology·Michal ShpilmanBerta Levavi-Sivan
Jan 15, 2008·General and Comparative Endocrinology·M YacobovitzA Gertler
Jun 17, 2011·Neuroendocrinology·Robert J DenverGraham C Boorse

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