Leptinegepegyleerde antagonist (mutant L39A/D40A/F41A)

Mono-pegylated (with 20 kDa PEG) recombinant human 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 recombinant human leptin antagonist runs on the SDS-PAGE as 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. Human pegylated recombinant leptin antagonist half-life in circulation after SC injection was over 20 hours. Human pegylated recombinant leptin antagonist was initially mutated, resulting in L39A/D40A/F41A mutant that was purified by proprietary chromatographic techniques according to Niv-Spector et al, Biochem. J 291;221-230 (2005) . and its pegylation was similar to that is described in Elinav et al. for mouse leptin antagonist see Endocrinology 150:3083-91 (2009) .

Lep; ob; obese

3952

P41159

NP_000221.1

NM_000230

Human

Human

PEG

AVPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQKVTGAAAIPGLHPILTLSKMDQTLAVYQQILTSMPSRNVIQISNDLENLRDLLHVLAFSKSCHLPWASGLETLDSLGGVLEASGYSTEVVALSRLQGSLQDMLWQLDLSPGC

It is recommended to reconstitute the lyophilized pegylated recombinant human pegylated 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)

> 98.0% as determined by Gel filtration and SDS-PAGE gel.

Recombinant human pegylated leptin antagonist is capable of inhibiting leptin-induced proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. Its in vitro activity is 6-8 fold lower than the non-pegylated human leptin antagonist but in vivo it has profound weight gain effect in mice (as compared to the non-pegylated human leptin antagonist), resulting mainly from increased food intake.

146

Lyophilized

Water

35.6 kDa

E. coli

AVPIQ