古细菌Pyrococcus furiosus嗜热α-淀粉酶基因在大肠杆菌中的表达  

Expression of a Hyperthermophilic α-Amylase of the Archaeon Pyrococcus furiosus in Escherichia coli

作　　者：沈微[1] 王正祥[1] 刘吉泉[1] 诸葛健[1] 

机构地区：[1]江南大学生物工程学院和教育部工业生物技术重点实验室

出　　处：《食品与发酵工业》2003年第3期10-14,共5页Food and Fermentation Industries

基　　金：教育部骨干教师资助计划(No.1696)

摘　　要：用PCR方法从嗜热古细菌Pyrococcusfuriosus的基因组DNA中扩增出胞外α 淀粉酶完整结构基因 ,插入表达载体pET2 8a中构建成质粒 pET amy(sig+) ,以质粒pET amy(sig +)为底物扩增出不含信号序列的α 淀粉酶成熟肽基因片断 ,获得质粒 pET amy(sig ) ,将重组质粒分别转化大肠杆菌BL2 1 (DE3)。在T7启动子和lac操纵子控制下 ,通过IPTG的诱导在重组大肠杆菌细胞内分别表达出含信号肽和不含信号肽的融合蛋白。其中不含信号肽的融合蛋白具有与P .furio sus产生的胞外α 淀粉酶相似的酶学性质 :最适pH为 5 .0 ,最适温度约为 95℃ ,在 1 2 1℃下热处理 1h酶活仍能保持 50 %以上 ;The structure gene encoding peptide of extracellular α amylase was amplified from the genome DNA of hyperthermophilic archaeon Pyrococcus furiosus by PCR. The recombinant plasmid pET amy(sig+) was constructed by inserting the amplified segment into expression vector pET28a, the gene fragment encoding peptide of the amylase without signal sequence was amplified from pET amy(sig+), and inserted into pET28a too. Both recombinant plasmid was used to transform E. coli BL21(DE3). Promoted and controlled by T7 promoter and lac operator,the fusion protein include His tag of the pET28a and the peptide of α amylase with or without signal peptide was expressed within the cell of recombinant E. coli BL21(DE3). The fusion protein without signal peptide has the similar enzymatic properties as the extracellular α amylase produced by Pyrococcus furious: it shows an enzymatic activity optimum at pH 5.0, and it's optimal temperature for enzymatic activity is about 95℃, more than 50% of it's initial enzymatic activity is still detectable after it was incubated at 121℃ for an hour; the fusion protein with signal peptide is inactive.

关 键 词：“Pyrococcus furiosus” 嗜热α-淀粉酶 基因 PCR方法 融合表达 大肠杆菌细胞 酶学性质 

分 类 号：Q786[生物学—分子生物学]