小肽多拷贝基因表达载体的构建及其高效表达(英文)

介绍一种快速、高效构建小肽多拷贝基因表达载体的策略 ,并构建了相应的表达载体pETE coT .用人工合成的编码 2 8个氨基酸残基的胸腺素α1基因为模型 ,采用限制酶EcoT14I识别序列CCAAGG为小肽基因两末端序列 ,利用其酶切后可产生非镜相对称粘性末端 ,一次连接反应就构建出一系列不同基因拷贝数的表达载体 ;在小肽基因两端分别引进编码FactorXa和羟胺蛋白切割位点的序列 ,表达出的融合蛋白可被FactorXa和羟胺剪切出不残留任何外源氨基酸的小肽 .不同拷贝数的小肽融合蛋白在大肠杆菌BL2 1(DE3)中均获得高效表达 .

A Series of pET-derived Vectors for High-level Expression of the Gene of Multiple Copies Smaller Peptide in E.coli

A rapid method for constructing a series of expression vectors carrying multiple copies smaller peptide gene was described. An artificial gene of thymosin α1 (28 amino acid residues) was used as a model to be cloned into a vector from pET32a ( + ). A unique EcoT14 Ⅰ (CCAAGG) cloning site was introduced into the modified pET32a ( + ) to allow unidirectional insertion of multiple coding sequences. For high-level expression, multiple copies of target gene were cloned as tandem repeats interspersing with two different sites, where the fusion protein could be cleaved with Factor Xa and hydroxylamine to release monomer peptide units remaining no any additional amino acid. The different copies of fusion protein were expressed at high-level in E. coli BL21 (DE3).