Shewanella piezotolerans WP3中sulfotransferase基因的克隆表达与活性研究

Cloning,Expression and Identification of Sulfotransferase from S. piezotolerans WP3

Shewanella piezotolerans WP3分离自西太平洋1914 m的深海沉积物中,是一株革兰氏阴性,耐冷、耐压且兼性厌氧的杆状细菌.初步研究证明海洋细菌中含有磺基转移酶(sulfotransferase,ST)活性.本实验中构建了WP3两个磺基转移酶基因的原核表达载体,在E.coli中表达并纯化了重组ST,通过底物的化学反应,证明两个重组磺基转移酶均具有转磺酶活性.研究结果为后续实验的WP3胞外寡糖的离体硫酸化修饰工作奠定了基础.

S.piezotolerans WP3 is psychrotolerant and piezotolerant deep sea bacterium with a broad pressure optimum, extending from atmospheric pressure to about 20 MPa. The sulfotransferase（ST） of the bacterium is studied in this paper. Two genes, Swp1579 and Swp 4147, encoding the STs were cloned by PCR method. The gene Swp 1579 contains an open reading frame （ORF） of 849 bp, encoding a 526 aa putative ST protein, while the ORF of Swp 4147 consists of 1707 bp, encoding 568aa WP3 arylsulfate sulfotransferase. The 900bp and 1700 bp PCR product is obtained and ligated to the pQE30 vector and pQE70 vector, respectively. Utilizing the IPTG treatment, the cloned ST genes was expressed in Escherichia coli M15. The enzymatic activity assays of recombination proteins showed both had ST activities.