Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans
Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans
复制标题
在浅青紫链霉菌中生产 C 端酰胺化重组鲑鱼降钙素
DOI：
10.1385/abab:110:2:113
复制
发表时间：
2003-08-01
影响因子：
3
通讯作者：
Li, Y
中科院分区：
文献类型：
--
作者：
Hong, B;Wu, BY;Li, Y
鲑鱼降钙素（sCT）是需要C-末端酰胺化以获得完全生物活性的许多生物活性肽之一。为了大规模生产完全生物活性的sCT，我们构建了变铅青链霉菌[pMSA]，一种工程链霉菌菌株。在表达载体中，甘氨酸延伸的sCT、酰胺化底物和大鼠α-酰胺化酶cDNA在pIJ 680中的来自Streptomyces fraudiaphh基因的强组成型启动子的控制下克隆。两者均通过使用melC 1的表达和分泌信号的重组菌株以分泌方式表达。将细胞外表达的重组sCT纯化至接近均一性，并通过酶免疫测定法进行表征，然后进行直接氨基末端测序。高效液相色谱法、基质辅助激光解吸电离-飞行时间质谱法和体内生物测定法证明纯化产物与合成标准品等同。因此，工程化的链霉菌菌株可以直接在培养物上清液中产生生物活性的、C-末端酰胺化的重组sCT。重组过程的简易性及其扩大规模的潜力使其适应sCT的生产需求，并且可以应用于需要C-末端酰胺化的其他生物活性肽。
Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat alpha-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.