洋葱伯克霍尔德氏菌L68邻苯二酚2,3-双加氧酶基因克隆、表达和纯化  

Cloning,Expression and Purification of Catechol 2,3-Dioxygenase from Burkholderia cepacia L68

作　　者：吕鹏[1] 刘大桅[1] 张长铠[1] 

机构地区：[1]山东大学微生物技术国家重点实验室,山东济南250100

出　　处：《食品与生物技术学报》2006年第3期11-14,共4页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(30370014)

摘　　要：采用自行设计的引物,从洋葱伯克霍尔德氏菌L68中PCR扩增得到phnE(邻苯二酚2,3-双加氧酶)基因,亚克隆到高表达载体pET 32a上,转入表达菌株中。经双向测序,证实构建过程中未出现突变。重组子经IPTG诱导后,可以表达有活性的重组双加氧酶。选择26℃进行重组蛋白诱导。薄层扫描显示,表达重组蛋白总量最高可占总蛋白的64.92%。利用金属螯合层析对重组蛋白进行了一步纯化,SDS-PAGE结果显示,重组蛋白纯度达到95%。Catechol 2,3-dioxygenase（C230） gene was amplified with the designed primers from the total DNA of Burkholderia cepaciaL68. The PCR product was double digested and connected with the pET 32a vector. The recombined vector was transformed to E. coli BL21 trxB （DE3）. After induced by 5 mM IPTG, the lysis of the transformants showed specific C230 activities. Three temperatures were tested to optimize the induce temperature, and 26℃ was selected as the optimal induce temperature. The total recombined C230 protein was about 64.92 % of the total cell proteins base on the thin layer scanning analysis. The recombined C230 was further purified with Ni-NTA His Bind Resins to electrophoretic homogeneity.

关 键 词：洋葱伯克霍尔德氏菌 邻苯二酚2 3-双加氧酶 克隆表达 金属螯合层析 

分 类 号：Q78[生物学—分子生物学] Q936