Certificate of Analysis & Data Sheet

Full‐length cDNA encoding two leptin sequences (tLepA and tLepB) were identified in tilapia (Oreochromis niloticus). The cDNAs of tLepA and tLepB were 486 bp and 459 bp in length, encoding proteins of 161 aa and 152 aa, respectively. The tLepB ‐expressing plasmid was transformed into E. coli and expressed as recombinant proteins upon induction with nalidixic acid, found almost entirely in insoluble inclusion bodies (IBs). The protein was solubilized, refolded and purified to homogeneity by anion‐exchange chromatography. More information can be found in Shpilman et al. (2014) General and Comparative Endocrinology 270, 74-85.

E. coli

White lyophilized (freeze-dried) powder.

The recombinant Tilapia B leptin was lyophilized from a concentrated (1.0 mg/ml) solution containing NaHCO3 at 1:2 salt:protein ratio.

It is recommended to reconstitute the lyophilized recombinant Tilapia leptin B 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 Tilapia leptin B although stable at room temperature for several weeks, should be stored desiccated below -18C. Upon reconstitution of recombinant tilapia leptins at > 0.1 mg/ml and up to 2 mg/ml and filter sterilization recombinant tilapia leptins can be stored at 4C for several weeks. At lower concentration of recombinant tilapia leptins addition of a carrier protein (0.1% HSA or BSA) is suggested. Please prevent freeze-thaw cycles.

The purity of recombinant tilapia leptins 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 first six N-terminal amino acids of recombinant Tilapia leptin B was determined and was found to be Ala-Leu-Leu-Thr-Lys-Gly.

The purified monomeric recombinant Tilapia leptin B (16K) consists of > 90% monomers as determined by gel-filtration chromatography.

Monomeric and dimeric Tilapia leptins were biologically active in promoting proliferation of BAF/3 cells stably transfected with the long form of human leptin receptor (hLepR), but their activity was four orders of magnitude lower than that of mammalian leptin. Furthermore, the Tilapia leptins were biologically active in promoting STAT‐LUC activation in COS7 cells transfected with Tilapia leptin receptor but not in cells transfected with human leptin receptor. Tilapia Leptin A was more active than Tilapia Leptin B.

Less than 0.1 ng/µg (IEU/µg) of recombinant Tilapia leptins.

Protein quantitation of recombinant Tilapia leptin B was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.19 for 1 mg/ml (tLepB) as the extinction coefficient for a 0.1% (1mg/ml) solution at pH 8.0. This value is calculated by the DNAman computer analysis program of protein sequences.

For research purposes only.