Protein Refolding And Pharmacodynamic Evaluation Of BNP Molecule With High Activity

Sodium uretic peptides are a group of molecules with similar structures and functions. They bind to specific receptors, lead to the release of signal molecules like guanylate cyclase, and thus reduce blood pressure and cardiac load. While rhBNP (recombinant human BNP) has been approved for marketing for several years, recent research questioned its effect in clinical use. Due to rhBNP has lots of secondary structures, we propose to prepare the one with high-activity, to support future formulation work.Rabbit isolated vascular perfusion experiment reveals that the chemically synthesised reduced BNP and E. coli-expressed BNP molecules don’t have obvious effects on blood vessel. This informs us that screening for the effective molecules with specific secondary structures is necessary to increase the activity. Chemically synthesised BNP and in-vitro protein refolding technology were used to obtain high activity BNP molecules. The in vitro activity was assessed through NPR-A binding and blood vessel assay and the in vivo activity was evaluated with zebrafish experiments. The potential formulation of this optimized BNP was obtained through the studying the activity of this peptide in different buffer systems.After screening9sets of pre-defined refolding conditions, we find that the solution containing Tris, NaCl, KCl, L-Arginine, GSG, GSSG and EDTA was the best one to get activated form of BNP molecules. The NPR-A binding activity of this refolded BNP is obviously better than the nesiritide produced by Chen Pep Inc. Zebrafish experiments indicate that refolded BNP has an obvious effect in improving Heart blood clot, through which we constructed a clear relationship between dosage and effect. Zebrafish experiments also reveal that optimised BNP molecules maintain its stable therapeutic activity at the pH of4to5in Citrate acid/Na2HPO4buffer system.Our study shows that it is very important to optimize and restrict the secondary structure during the development of peptide drugs. Our findings provide feasible and pragmatic supports to peptide pharmaceutic formulation research.