Large-scale preparation and characterization of non-pegylated and pegylated superactive ovine leptin antagonist

Leonora Niv-Spector, Michal Shpilman, Yves Boisclair, Arieh Gertler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

19 Scopus citations

Abstract

Superactive ovine leptin antagonist (SOLA) was prepared by rational mutagenesis of the ovine leptin antagonist L39A/D40A/F41A mutant prepared previously in our lab by mutating wild type leptin to D23L/L39A/D40A/F41A. SOLA was expressed in Escherichia coli as insoluble inclusion bodies, refolded and purified to homogeneity (as evidenced by SDS-PAGE and analytical gel filtration) by ion-exchange chromatography. The purified protein was mono-pegylated at its N terminus by 20-kDa linear pegylation reagent. The D23L mutation resulted in ca. 5- to 6-fold increased affinity toward soluble human leptin binding domain and 6- to 8-fold increased inhibitory activity in two different in vitro bioassays. This increase was similar, though not identical, to our previous results with superactive mouse and human leptin antagonists. Pegylation decreased overall activity by 5- to 8-fold, but as shown previously for superactive mouse leptin antagonist, the prolonged half life in the circulation will likely result in higher activity in vivo. As amino acids 6-31 (VQDDTKTLIKTIVTRINDISHTQSVS), making up a main part of the first α-helix, are identical in human, mouse, rat, ovine, bovine and pig leptins, we anticipate that D23L mutations of the respective leptins will result in similar increases in affinity and consequent activity of other leptin antagonists.

Original languageEnglish
Pages (from-to)186-192
Number of pages7
JournalProtein Expression and Purification
Volume81
Issue number2
DOIs
StatePublished - Feb 2012

Keywords

  • Bioassay
  • Leptin antagonist
  • Ovine
  • Recombinant
  • Superactive

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