β-琼脂糖酶ⅠDagA大肠杆菌表达系统的筛选  

Screening of the β-agaraseⅠ DagA Expression Systems in Escherichia coli

作　　者：周雁胜[1] 王保莉[1] 曲东[2] 

机构地区：[1]西北农林科技大学生命科学学院,陕西杨凌712100 [2]西北农林科技大学资源与环境学院,陕西杨凌712100

出　　处：《西北农业学报》2008年第6期271-275,共5页Acta Agriculturae Boreali-occidentalis Sinica

摘　　要：通过PCR技术扩增出了873 bp的dagA及813bp的去除信号肽的dagA(▽)编码序列,dagA与NCBI公布的Pseudoalteromonas atlanticaβ-琼脂糖酶ⅠdagA序列一致性达到99.8%,dagA(▽)一致性达到100%。构建pET21a-dagA和pET21a-dagA(▽)表达载体,分别转化BL21(DE3)、Origami B(DE3)、ER2566宿主菌,共表达分子伴侣DsbC和FkpA。采用SDS-PAGE分析了构建的多个大肠杆菌表达系统中β-琼脂糖酶ⅠDagA的表达量及表达方式,获得了重组DagA高分泌性表达菌株ER2566-pET21a-dagA(▽)-FkpA。The coding region of dagA gene and dagA（▽） gene without signal peptide were cloned by PCR and the length were 873bp and 813bp respectively. The similarity between the coding region of dagA gene and Pseudomonas atlantica β-agarase Ⅰ dagA from NCBI is 99.8%, and of dagA（▽ ） is 100%. Both of the genes were co-expressed with molecular chaperones by construction of express vector of pET21a-dagA and pET21a-dagA（ ▽ ）, transforming to different hosts which are BL21（DE3）, Origami B（DE3）and ER2566. The recombinant strain of ER2566-pET21a-dagA（ ▽ ）-FkpA was isolated as a high effective secretory expression strain after analyzing the character and quantity of the expression realized by different E. coli expression systems through SDS-PAGE electrophoresis.

关 键 词：β-琼脂糖酶ⅠDagA 信号肽 分子伴侣 分泌表达 

分 类 号：Q786[生物学—分子生物学]