The Study Of In Vitro Stability And Bioactivities Of Recombinant Polypeptide RMBAY

VPAC2 is a co-receptor shared by Pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP), which played important roles in energy metabolism and immunoregulation. A recombinant named RMBAY was designed and prepared using intein-mediated purification system as a novel agonist for VPAC2. The goal of the research is to improve the stability in vitro of the RMBAY.A C-terminal thioester of the target peptide was produced as an intermediate product, which was unstable and hydrolysed easily to release free terminus. In order to induce directional hydrolyzation to release thio- carboxylic acid, the C-terminal thioester of the target peptide was dialysed in multiple conditions with different pH values or with different thiol such as NaHS or MESNA. The results of SDS-PAGE and Mass Spectrometry showed both the alkalinity and thiol help the directional hydrolysis of C-terminal thioester to release thio- carboxylic acid. And MESNA(50mM) played a stronger effect on promoting the stability in vitro of the target peptide than NaHS.The bioactivity assay of RMBAY on ruducing the plasma glucose revealed that the RMBAY not only had an effect on ruduing the plasma glucose by promoting the glucose dependent insulin release, but also improve the serum glucose tolerance in NIH mice.The model of passive cutaneous anaphylaxis (PCA) on NIH mouse were established and used for the bioactivity assay of RMBAY. The results showed that RMBAY (5nmol/kg) displayed a suppressive effect(0.01