Certificate of Analysis & Data Sheet

The first biologically active recombinant fish leptin was prepared according to the sequence published by Kurokawa et al. (2005)Peptides 26, 745-750 in two forms: monomer and covalent dimer. MS analysis revealed molecular masses of 15,291 and 30,585 Da, close to the theoretical values of 15,270 and 30,540 Da. CD spectra revealed high similarity to mammalian leptins. Other details of its preparation will be soon published by Yacobovitz et al , General and Comparative Endocrinology (2008) 156(1):83-90.

E. coli

White lyophilized (freeze-dried) powder.

The recombinant fish leptin was lyophilized from a concentrated (0.85mg/ml) solution with 0.003mM NaHCO3.

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

The purity of recombinant fish leptin is greater than 98.0% as determined by:
(a) Gel filtration analysis.
(b) Analysis by reducing and non-reducing SDS-PAGE gel.
(c) RP-chromatography

The sequence of the first five N-terminal amino acids of recombinant fish leptin was determined and was found to be Ala-Leu-Pro-Gly-Ala.

The purified monomeric recombinant fish leptin (16K) consists of > 97% monomers as determined by gel-filtration chromatography.
The covalent dimer contains less than 2% monomers.

PLR’s recombinant fish leptin is capable of inducing proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor. The affinity of human leptin receptors is considerably lower campared to mammalian leptins.

Less than 0.1 ng/µg (IEU/µg) of recombinant fish leptin

Protein quantitation of recombinant fish leptin was carried out by UV spectroscopy at 280 nm using the absorbency value of 1.28 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).

The recombinant fish leptin is offered by PLR for laboratory research