Certificate of Analysis & Data Sheet

Recombinant protein - human leptin N82K mutant was produced by site specific mutagenesis. It consists of one polypeptide chain containing 146 amino and additional Ala at N-terminus acids and having a molecular mass of ~ 16 kDa., Human leptin mutant was purified by proprietary chromatographic techniques, see Niv-Spector et al. (2010) Mol Genet Metab. 100(2):193-7. It is devoid of biological activity and mey serve for control experiments.

Escherichia coli

White lyophilized (freeze-dried) powder.

The recombinant protein was lyophilized from a concentrated (1mg/ml) solution with 0.0045mM NaHCO3.

It is recommended to reconstitute the lyophilized recombinant human leptin N82K mutant in sterile water or 0.4% NaHCO3 adjusted to pH 8-9, not less than 100µg/ml, which can then be further diluted to other aqueous solutions.

Lyophilized recombinant human leptin N82K mutant although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution at > 0.1 mg human leptin per ml and up to 2 mg and filter sterilization recombinant human leptin N82K mutant can be stored at +4C or even room temperature for several weeks. At lower concentration addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

Purity of recombinant human leptin N82K mutant is greater than 98.0% as determined by:
(a) Gel filtration analysis of the recombinant protein.
(b) Analysis by reducing and non-reducing SDS-PAGE gel.

The sequence of the first five N-terminal amino acids of recombinant human leptin N82K mutant was determined and was found to be Ala-Val-Pro-Ile-Gln.

The purified recombinant human leptin (16K) N82K mutant consists of > 98% monomers as determined by gel-filtration chromatography.

PLR’s recombinant human leptin N82K mutant is less than 0.1% active active as evidenced by inducing proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. This abolishment of activity results from drastically reduced affinity toward leptin receptor.

The endotoxin content of recombinant human leptin is less than 0.05 ng/µg (0.5 IEU/µg).

Protein quantitation in recombinant human leptin N82K mutant 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) solution at pH 8.0. This value is calculated by the PC GENE computer analysis program of protein sequences (IntelliGenetics).

Recombinant human leptin N82K mutant is offered by PLR for laboratory research as a control of inactive leptin.