几丁质结合蛋白基因克隆、表达与纯化

Cloning, expression and purification of chitin-binding protein gene

该研究通过聚合酶链反应(PCR)方法从假交替单胞菌属(Pseudoalteromonas sp.)DL-6菌株中成功克隆了几丁质结合蛋白基因。PCR测序结果表明,该基因全长1 596 bp,编码531个氨基酸,其理论分子质量为58.517 ku,等电点(p I)4.35,命名为CBP58(GenBank登录号KF234016)。结构域分析结果表明,该蛋白包括1个33家族碳水化合物结合模块(CBM),2个类型3几丁质结合域(Cht BDs);用Insight II 2005软件以同源建模的方法构建CBP58蛋白CBM33结构域的三维结构模型,Ramachandram图谱检测和三维结构评估显示模型结构合理,整体相容性较为可信;将CBP58基因构建到p ET23b载体,并转化至大肠杆菌(Escherichia coli)BL21(DE3)中诱导表达;利用镍柱亲和层析纯化获得重组蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测显示目的蛋白可溶表达,为后期几丁质结合蛋白(CBP)的生化性质表征奠定理论基础。

Chitin-binding protein gene was cloned successfully from Pseudoalteromonas sp. DL-6 by polymerase chain reaction （PCR）. The PCR se- quencing results indicated that the gene was 1 596 bp in length, and encoded 531 amino acid, and its theoretical molecular mass and isoelectric point （pI） was 58.517 ku and 4.35, respectively. It was named as CBP58 （Genebank no. KF234016）. The structural domain analysis results showed that the chitin binding protein contained one 33 family carbohydrate-binding modules （CBM）, two copies of chitin-binding domain type 3 classified （ChtBDs）. The three-dimensional structure model of the homology structure of CBM33 structural domain of the CBP58 protein was built by homologous model- ing on Insight I12005. The diagram of Ramachandram plot and Verify-3D showed that the structure of model was reasonable and the overall compat- ibility of this model was credible. The CBP58 gene was constructed into the pET23b vector and then transformed into Escherichia coli BL21 （DE3） to be induced for expression. The recombinant proteins were purified by Ni-NTA resin. CBP58 was expressed successfully by the analysis of sodium dodecyl sulphate-polyacrylamide gel electrophoresis （SDS-PAGE）. The study laid a theoretical foundation for the biochemical characterization of the CBP.