Certificate of Analysis & Data Sheet

Mono-pegylated mouse super leptin antagonist (with 20 kDa PEG) 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 mouse leptin antagonist runs on the SDS-PAGE as a 55 kDa protein and in gel-filtration on Superdex 200 as over 200 kDa protein. Pegylated recombinant super mouse leptin antagonist half-life in circulation after SC injection was over 20 hours. Super mouse leptin antagonist (SMLA) was initially mutated, resulting in D23L/L39A/D40A/F41A mutant that was purified by proprietary chromatographic techniques. Its pegylation is similar to that described in Elinav et al. Endocrinology 150:3083-91 (2009) for pegylated recombinant mouse leptin antagonist.

Escherichia coli.

White lyophilized (freeze-dried) powder.

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

It is recommended to reconstitute the lyophilized pegylated recombinant super mouse 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 super mouse leptin antagonistal though stable at room temperature for several weeks, should be stored desiccated below - 20C. Upon reconstitution of pegylated recombinant super mouse leptin antagonist at > 0.1 mg/ml and up to 2 mg/ml and filter sterilization pegylated recombinant super mouse leptin antagonist mutant 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 pegylated recombinant super mouse 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 pegylated recombinant super mouse leptin antagonist could not be determined indicating that the PEG moiety is attached to the N-terminus.

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

PLR’s pegylated recombinant super mouse leptin antagonist is capable of inhibiting leptin-induced proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. Pegylated recombinant super mouse leptin antagonist in vitro activity is 6-8 fold lower than the non-pegylated pegylated recombinant super mouse leptin antagonist but in vivo it has profound weight gain effect (as compared to the non-pegylated antagonist), resulting mainly from increased food intake. Its in vivo activity compared to that of PEG-MLA is 9-27 fold higher.

The endotoxin content of recombinant super mouse leptin antagonist is less than 0.01 ng/µg (0.1 EU/µg of protein) of PEG-SMLA

Protein quantization of recombinant super mouse 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 super mouse 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.