Certificate of Analysis & Data Sheet

Mono-pegylated (with 20 kDa PEG)recombinant 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 it runs on the SGS-Page as 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. Ovine leptin was initially mutated, resulting in L39A/D40A/F41A mutant that was purified by proprietary chromatographic techniques according to Niv-Spector et al (2005) Biochemical Journal, 291, 221-230 and its pegylation was similar to that of mouse leptin antagonist is described in Elinav et al. Endocrinology (2009)150, 3083-3091.

Escherichia coli.

White lyophilized (freeze-dried) powder.

The recombinant ovine pegylated leptin antagonist was lyophilized from a concentrated (0.65mg/ml) solution with 0.003mM NaHCO3.

It is recommended to reconstitute the lyophilized recombinant ovine 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 recombinant ovine pegylated leptin antagonist although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution at > 0.1 mg/ml and up to 2 mg/ml and filter sterilization recombinant ovine 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 of recombinant ovine pegylated leptin antagonist addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

The purity of recombinant ovine pegylated leptin antagonist is greater than 95.0% 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 ovine pegylated leptin antagonist could not be determined indicating that the PEG moiety is attached to the N-terminus.

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

PLR’s recombinant ovine 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 antagonist but in vivo it has profound weight gain effect (as compared to the non-pegylated antagonist like in mouse leptin antagonists), resulting mainly from increased food intake.

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

Protein quantization of recombinant ovine pegylated leptin antagonist was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.200 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 recombinant ovine 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.