Fish gilthead seabream (Sparus aurata) IGF-I - Cat no. GHP-10 GENE ID not reported

Gilthead seabream (Sparus aurata) insulin-like growth factor-I (gsIGF-I) cDNA coding the mature protein was expressed in Escherichia coli. The expressed protein contained within the inclusion-body pellet was solubilized and purified to over 98% purity, as a monomeric methionyl-gsIGF-I. Amino acid composition and N-terminal sequence confirmed the identity to be the predicted protein. For reference see Fine et al., J Endocrinol. 1997 53:139-150.

E. coli.

 

 

White lyophilized (freeze-dried) powder

The protein was lyophilized from a concentrated (1mg/ml) solution with 0.02% NaHCO3.

It is recommended to reconstitute the lyophilized gsIGF-I in sterile 0.4% NaHCO3 adjusted, not less than 100µg/ml, which can then be further diluted to other aqueous solutions.

Lyophilized gsIGF-I although stable at room temperature for several weeks, should be stored desiccated below -18 C. Upon reconstitution at > 0.1 gsIGF-I mg/ml and up to 2 2 mg/ml and filter sterilization gsIGF-I can be stored at +4C.

Greater than 98.0% as determined by:
(a) Analysis by reducing and non-reducing SDS-PAGE gel.
(b) Gel-filtration chromatography under non denaturing conditions.

The sequence of the first ten N-terminal amino acids was determined and was found to be Met-Ser-Pro-Glu-Thr-Leu-Cys-Gly-Ala-Glu.

The purified gsIGF-I consists of > 97% monomers as determined by gel-filtration chromatography.

Binding assays of the 125I-gsIGF-I to gilthead seabream or carp (Cyprinus carpio) sera resulted in high specific binding, indicating the existence of one or more IGF-binding proteins. In binding experiments to crude gilthead seabream brain homogenate, using human (h) IGF-I as a ligand, the respective IC50 value of hIGF-I was about fourfold lower than that of gsIGF-I. Recombinant gsIGF-I exhibited mitogenic activity in a mouse mammary gland-derived MME-L1 cell line which was approximately 200-fold lower than that of hIGF-1. Binding experiments to intact MME-L1 cells suggests that this difference most likely results from a correspondingly lower affinity for IGF-I receptor in these cells. In contrast, the activities of gsIGF-I and hIGF-I measured by 35S uptake by gill arches from the goldfish (Carassius auratus) were identical, indicating that the recombinant gsIGF-I is biologically active.

Less than 0.1 ng/µg (IEU/µg) of Gilthead Seabream IGF-I

Protein quantization was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.60 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.

This material is offered by PLR for laboratory research