Leptine gepegyleerde superantagonist (mutant D23L/L39A/D40A/F41A)

Mono-pegylated (with 20 kDa PEG) recombinant super active ovine leptin antagonist is one polypeptide chain containing 146 amino, an additional Ala at N-terminus and one molecule of PEG 20 kDa at its N-terminus, having an expected molecular mass of ~ 35.6 kDa as determined by MS. However due to enlarged hydrodymanic volume pegylated recombinant super active ovine leptin antagonist runs on the SDS-PAGE as 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. The half-life in circulation of pegylated recombinant super active ovine leptin antagonist after SC injection was over 20 hours. Recombinant super active ovine leptin antagonist was initially mutated, resulting in D23L/L39A/D40A/F41A mutant that was purified by proprietary chromatographic techniques. The pegylation of recombinant super active ovine leptin antagonist is similar to that described in Elinav et al. Endocrinology 150:3083-91 (2009) for pegylated recombinant super active mouse leptin antagonist

Lep; ob; obese

443534

G8BLB2

XP_027824581.2

XM_027968780.2

Ovine

Ovine

PEG

AVPIRKVQDDTKTLIKTIVTRINLISHTQSVSSKQRVTGAAAAPGLHPLLSLSKMDQTLAIYQQILASLPSRNVIQISNDLENLRDLLHLLAASKSCPLPQVRALESLESLGVVLEASLYSTEVVALSRLQGSLQDMLRQLDLSPG

It is recommended to reconstitute the lyophilized pegylated recombinant super active ovine leptin antagonist in sterile water or sterile 0.4% NaHCO3 adjusted to pH 8-9, not less than 100µg/ml, which can then be further diluted with other aqueous solutions.

< 0.1 ng/µg of protein (< 1EU/µg)

> 95.0% as determined by Gel filtration and SDS-PAGE gel.

Pegylated recombinant super active ovine leptin antagonist is capable of inhibiting leptin-induced proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The in vitro activity of pegylated recombinant super active ovine leptin antagonist is 6-8 fold lower than the non-pegylated antagonist but is 15 fold higher as compared to pegylated recombinant super active ovine leptin antagonist

146

Lyophilized

Water

35.6 kDa

E. coli

AVPIR