Certificate of Analysis & Data Sheet

Mono-pegylated (with 20 kDa PEG)recombinant mouse leptin mutant D23L is an 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 mouse pegylated leptin runs on the SGS-Page as 48 kDa protein and in gel-filtration on Superdex 200 as over 100 kDa protein. Recombinant mouse pegylated leptin mutant D23L half-life in circulation after SC injection was over 20 hours. Recombinant mouse leptin mutant D23Lwas purified by proprietary chromatographic techniques similarly to Salomon et al (2006) Protein Expression and Purification 47, 128–136 and then pegylated.

Escherichia coli.

White lyophilized (freeze-dried) powder.

The recombinant mouse pegylated leptin was lyophilized from a concentrated (0.65mg/ml) solution with 0.003mM NaHCO3 and the powder contains 35 to 40% protein.

It is recommended to reconstitute the lyophilized recombinant mouse pegylated leptin 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 mutant D23L although stable at room temperature for several weeks, should be stored desiccated below -20C. Upon reconstitution at > 0.1 recombinant mouse pegylated leptin mg/ml and up to 2 mg/ml and filter sterilization recombinant mouse pegylated leptin muatnt D23L 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 addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

The purity od recombinant mouse pegylated leptin is greater than 99.0% 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 could not be determined indicating that the PEG moiety is attached to the N-terminus.

The purified recombinant mouse pegylated leptin consists of > 95% monomers as determined by gel-filtration chromatography.

PLR’s recombinant mouse pegylated leptin mutant D23L is capable of stimulatng proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. Its in vitro activity is only slightly lower than the non-pegylated recombinant mouse leptin but in vivo it has profound weight reducing effect (as compared to the non-pegylated recombinant mouse leptin ), resulting mainly from reduced food intake.

Less than 0.05 ng/µg (0.5 IEU/µg) of recombinant mouse pegylated leptin .

Protein quantization of recombinant mouse pegylated leptin mutant D23L 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 mouse pegylated leptin 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.