별불가사리(Patiria pectinifera)에서 분리한 Orexin A-related peptide의 수율 향상을 위한 연구 및 근육 활성 조사

Abstract

Study to improve the production yield of Orexin A-related peptide isolated from starfish (Patiria pectinifera) and investigation of it's myoactivity

In young Noh

School of Marine and Fisheries Life Science (Major in Biotechnology), Pukyong National University

Abstract Orexin A is a neuropeptide known to be involved in stabilizing arousal, increasing appetite, and interacting with other neurotransmitters. The orexin A-related peptide used in this study was purified from the tube feet of the starfish (Patiria pectinifera), and this peptide exhibited muscle contractile activity. The molecular weight and the primary structure of Orexin A-related peptide determined by LC/MS & cDNA cloning showed that the peptide has a molecular weight of 3,466Da and is composed of 33 amino acids including 6 cysteines that form 3 disulfide bonds. Orexin A-related peptide was produced in a heterologous expression system using the fusion expression vector pET-28a(+)-TrxA. The produced recombinant Orexin A-related peptide (r[Leu33]PpOrxA) was cleaved by CNBr treatment at the C-terminus of Met. The recombinant Orexin A-related peptide (r[Leu33]PpOrxA) thus produced has a -OH group at the C-terminus. On the other hand, native orexin A-related peptide that exists in nature have an amidated C-terminus. Therefore, the amidation process was performed using carboxypeptidase-Y (CPD-Y) and Met-NH2∙HCl. In addition, the optimal conditions for obtaining the maximum production yield of recombinant Orexin A-related peptide-Met-NH2 (rPpOrxA) by changing the amounts of pH, CPD-Y and DMF in the amidation process were investigated. The changes in pH affected the production yield. It was confirmed that the production yield was the highest at pH 6 among the three conditions (pH 6, 7, 8). Moreover, it was confirmed that the amount of CPD-Y also affects the production yield. On the other hand, the change in DMF did not cause a significant change in the production yield of the peptide. RT-qPCR was performed to determine the transcriptional expression level of the Orexin A-related peptide by tissue, and it was confirmed that the highest expression level was found in the radial nerve cord among various tissues of the starfish. The muscle contractile activity of the produced rPpOrxA was investigated. Muscle contractile activity by rPpOrxA was confirmed in three tissues (apical muscle, cardiac stomach, tube feet) of starfish. rPpOrxA showed the most potent contractile activity on apical muscle preparations among the three tissue preparations.