Certificate of Analysis & Data Sheet

Mono-pegylated (with 20 kDa PEG) recombinant mouse 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 mouse pegylated leptin antagonist runs on the SGS-Page as 48 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. Mouse pegylated leptin antagonist half-life in circulation after SC injection was over 20 hours.. Mouse leptin antagonist was initially mutated, resulting in L39A/D40A/F41A mutant that was purified by proprietary chromatographic techniques according to Salomon et al (2006) Protein Expression and Purification 47, 128–136 and its pegylation is described in Elinav et al. Endocrinology 150:3083-91 (2009).

Escherichia coli.

White lyophilized (freeze-dried) powder.

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

The recombinant mouse pegylated leptin antagonist was lyophilized from a concentrated (0.65mg/ml) solution with 0.003mM NaHCO3. It is recommended to reconstitute the lyophilized recombinant mouse 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 mouse pegylated leptin antagonist although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution at > 0.1 recombinant mouse pegylated leptin antagonist mg/ml and up to 2 mg/ml and filter sterilization recombinant mouse 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 mouse pegylated leptin antagonist addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

The purity of recombinant mouse pegylated leptin antagonist is more 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 mouse pegylated leptin antagonist could not be determined indicating that the PEG moiety is attached to the N-terminus.

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

PLR’s recombinant mouse pegylated 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 the pegylated recombinant mouse leptin antagonistis 6-8 fold lower than the non-pegylated antagonist but in vivo it has profound weight gain effect (as compared to the non-pegylated recombinant mouse leptin antagonist), resulting mainly from increased food intake.

The endotoxin content of pegylated recombinant mouse leptin antagonist is less than 0.05 ng/µg (IEU/µg).

Protein quantization of recombinant mouse pegylated leptin antagonist was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.201 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 mouse 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.