Certificate of Analysis & Data Sheet

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).

Escherichia coli.

White lyophilized (freeze-dried) powder

The recombinant protein was lyophilized from a concentrated (0.65mg/ml) solution with 0.003mM NaHCO3.

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.

Lyophilized pegylated recombinant human pegylated leptin antagonist although stable at room temperature for several weeks, should be stored desiccated below -20C. Upon reconstitution at > 0.1 pegylated human pegylated leptin antagonist mg/ml and up to 2 mg/ml and filter sterilization human pegylated leptin antagonist can be stored at 4C or even room temperature for several weeks making it suitable for long term infusion studies using osmotic pumps. At lower concentration addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

The purity of recombinant human pegylated leptin antagonist is greater than 98.0% as determined by:
(a) Gel filtration analysis.
(b) Analysis by reducing and non-reducing SDS-PAGE gel.

The sequence of the N-terminal amino acids of recombinant human pegylated leptin antagonist could not be determined indicating that the PEG moiety is attached to the N-terminus.

The purified recombinant human pegylated leptin antagonist consists of > 90% monomers as determined by gel-filtration chromatography.

PLR’s 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.

Less than 0.05 ng/µg (IEU/µg) of recombinant human leptin antagonist.

Protein quantitation of recombinant human pegylated leptin antagonist was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.87 as the extinction coefficient for a 0.1% (1mg/ml) protein solution at pH 8.0. This value is calculated by the PC GENE computer analysis program of protein sequences (IntelliGenetics).

PLR's human recombinant pegylated leptin antagonist is furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals. It is for Research Purposes Only Commercial Use has been Licensed Exclusively to a Third Party.