大鼠α-酰胺化酶(α-AE)在大肠杆菌中的表达、纯化及活性研究

α-酰胺化是神经和内分泌系统中许多生物活性肽重要的翻译后加工过程，C末端α酰胺基团的存在对于许多生物活性肽的生物活性极为重要。本研究通过PCR扩增获得了编码大鼠α-酰胺化酶的基因，以pET-30a为载体，重组质粒pET-A转化至 E.coli BL21成功表达了α-酰胺化酶。采用Ni2+-NTA亲和层析纯化重组蛋白。该蛋白具有催化三肽底物((Dns-Tyr-Val-Gly)成为酰胺化二肽 (Dns-Tyr-Val-NH2)的酶活性，这表明重组蛋白是α-酰胺化酶，有可能用于生物活性肽的酰胺化研究。

Purification and Characterization of α-AE Protein Expressed in E. coli

Many bioactive peptides from neural and endocrine tissue are amidated at C-terminals, which is essential for their activities. The α-amide comes from post-translational modification that is catalyzed by α-AE (α-amidating enzyme) or PAM (pepdilylglycine α-amidating monooxygenase). In this paper, the gene encoding α-AE was amplified with PCR and cloned into the plasmid pET-30a. After the recombinant plasmid pET-A was transformed into E.coli BL21, the α-AE was expressed and purified by the Ni2+ affinity chromatography, which has the ability catalyzing Dns-Tyr-Val-Gly to Dns-Tyr-Val-NH2. It identified that the recombinant protein producing by E.coli BL21 is α-AE, which will benefit for studies of amidation at the C-terminals of peptides.