protein

Rat growth hormone antagonist (G119R)

GHP-33

Recombinant rat GH-22K G119R produced in E. coli is a single, non-glycosylated, polypeptide chain containing 191 amino acids and having a molecular mass of 22 kDa. It is mutated in single amino acid (G119R) according to similar bovine GH mutein described by Chen et al. (1991), Molecular Endocrinology 5:1845–1852. Rat GH antagonist was purified by proprietary chromatographic techniques.

Escherichia coli

Sterile-filtered white lyophilized (freeze-dried) powder.

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

It is recommended to reconstitute the lyophilized rGH G119R in 0.4% NaHCO3 or water adjusted to pH 9, not less than 100µg/ml, which can then be further diluted to other aqueous solutions, preferably in a presence of a carrier protein such as BSA or similar.

Lyophilized rGH G119R although stable at room temperature for at least two weeks, should be stored desiccated below -18C. Upon reconstitution and filter sterilization oGH can be stored at 4C, pH 9 for up to 4 weeks. For long term storage and more diluted solutions it is recommended to add a carrier protein (0.1% HSA or BSA). Addition of carrier protein should be avoided in in vivo experiments to prevent immunic response, Please prevent freeze-thaw cycles.

Greater than 98.0% as determined by: (a) Analysis by RP-HPLC. (b) Gel filtration at non denaturing conditions using 25 nM Tris-HCl + 300 nM NaCl, pH 8. (c) Analysis by reducing and non-reducing SDS-PAGE Silver Stained gel.

The sequence of the first five N-terminal amino acids was determined and was found to be Ala-Phe-Pro-Ala-Met.

Less than 2% as determined by gel filtration chromatography

PLR’s rGH G119R acts as antagonist using an in vitro bioassay in PDF-P1 3B9 cells stably transfected with rabbit GH receptors. It is capable of forming a 1:1 complex with the recombinant ovine growth hormone receptor extracellular domain (ECD)and binds to this ECD with affinity similar the the wild type rGH.

Less than 0.05 ng/µg (IEU/µg) of rat GH G119R.

Protein quantitation was carried out by UV spectroscopy at 280 nm using the absorbency value of 0.69 as the extinction coefficient for a 0.1% (1mg/ml) solution. This value is calculated by the DNAman analysis program of protein sequences.

PLR's recombinant rat growth hormone antagonist can be used for research purpose only.