人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达

Couple Production of Human Calcitonin and Rat Peptidylglycine αamidation Monooxygenase in Insect Cells

Human calcitonin (hCT) is a 32 amino acid peptide hormone that requires C-terminal amidation for full biological activity. Calcitonin has important physiological function in vivo. We describe the couple expression of a synthesized modified human calcitonin(hmCT) gene fused with glutathione-S-transferase and rat peptidylglycine alpha-amidation monooxygenase (PAM) in insect cells infected by recombinant baculovirus GSTCT/PAM. Using Western blotting against hmCT or rat PAM, the GSThmCT fusion protein had been identified as well as the PAM. Following affinity chromatography with glutathione agarose column, the GSThmCT fusion protein produced by insect cells was purified. The purified fusion protein was also interacted with antibody against hmCT. The couple expression of a modification enzyme and its substrate in eucaryotic expression system may be used for producing other biological activity peptides.