Purification and characterization of recombinant pufferfish (Takifugu rubripes) leptin

M. Yacobovitz, G. Solomon, E. E. Gusakovsky, B. Levavi-Sivan, A. Gertler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations

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 50-100 mg from 5 L 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 ∼15 kDa following SDS-PAGE in the presence of reducing agent, whereas the dimer showed one band of ∼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 °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.

Original languageAmerican English
Pages (from-to)83-90
Number of pages8
JournalGeneral and Comparative Endocrinology
Volume156
Issue number1
DOIs
StatePublished - 1 Mar 2008
Externally publishedYes

Bibliographical note

Funding Information:
The authors thank Prof. Joe O’Neil at the Department of Chemistry, University of Manitoba, for use of the spectropolarimeter and Dr. Jean Djiane from INRA for the anti-leptin Ab. We also thank Mr. Gopal Savjani (Savjani Inc., Houston, TX, USA) for his generous support of Michal Yacobovitz’s fellowship.

Keywords

  • Biological activity
  • Leptin
  • Pufferfish
  • Recombinant

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