pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis
pAM401-based shuttle vectors that enable overexpression of promoterless genes and one-step purification of tag fusion proteins directly from Enterococcus faecalis
复制标题
DOI：
10.1128/aem.67.3.1262-1267.2001
复制
发表时间：
2001-03-01
影响因子：
4.4
通讯作者：
Ike, Y
中科院分区：
文献类型：
--
作者：
Fujimoto, S;Ike, Y
利用大肠杆菌上的巴卡A启动子和核糖体结合位点构建了两个新型粪肠球菌-大肠杆菌穿梭载体。构建了粪肠球菌质粒pPD 1，分别命名为pMGS 100和pMGS 101。设计pMGS 100在E.大肠杆菌和粪肠球菌，并编码巴卡启动子，随后是克隆位点和终止密码子。pMGS 101设计用于过表达和纯化与在羧基末端由9个氨基酸组成的Strep标签融合的克隆蛋白。Strep标签为克隆的蛋白质提供了对固定化链霉亲和素的亲和力，这有助于蛋白质纯化。我们从大肠杆菌中克隆了一个无启动子的β-半乳糖苷酶基因. coli，克隆了大肠杆菌的traA基因。粪肠杆菌质粒pAD 1插入载体中，分别检测基因表达和蛋白纯化。β-半乳糖苷酶在E. coli和E.粪菌水平分别为10（3）和10米勒单位。通过将pAD 1 traA克隆到pMGS 101中，可以直接从粪肠球菌或大肠杆菌的粗裂解物中纯化该蛋白。大肠杆菌与固定化链霉亲和素基质通过一步亲和层析。通过DNA相关蛋白标签亲和层析方法，使用从大肠杆菌和大肠杆菌制备的裂解物证明TraA结合DNA的能力。过表达TraA的粪肠球菌。结果表明，该载体可用于调控基因的过表达和顺反分析，以及大肠杆菌蛋白质的纯化和共纯化。粪大肠杆菌的DNA结合分析、翻译起始位点的确定以及其它需要从E.粪便。
Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed, The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E.faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless beta -galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. beta -Galactosidase was expressed in E. coli and E. faecalis at levels of 10(3) and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E.faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E, coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.