嗜热厌氧乙醇杆菌调控因子P_(adhE)-1的分离、重组表达及其与adhE基因启动子的结合  

Expression and Identification of the P_(adhE)-1 from Thermoanaerobacter ethanolicus JW200 and Its Binding with adhE Promoter in vitro

作　　者：裴建军[1] 周庆[2] 曲威[2] 邵蔚蓝[1,2] 

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]南京师范大学生命科学学院,江苏南京210046

出　　处：《微生物学通报》2010年第10期1501-1505,共5页Microbiology China

基　　金：国家自然科学基金项目(No.30900008);江苏省高校自然科学重点项目(No.09KJA180003)

摘　　要：嗜热厌氧乙醇杆菌发酵是生产纤维素乙醇的有效途径,而嗜热厌氧乙醇杆菌醛/醇脱氢酶(AdhE)是其乙醇代谢途径中的关键酶。以adhE启动子区域的DNA序列(TRRadhE)作为诱饵,通过制备核酸亲和层析柱,利用调控因子与DNA特异性结合的特点,得到调控因子PadhE-1。经过蛋白测序和NCBI数据库的比对,发现其N端序列与Thermoanaerobacter pseudethanolicus39E的LacI家族转录调控因子(GenBank accession No.YP_001665770)有90%的同源性。根据同源性设计引物,从嗜热厌氧乙醇杆菌JW200基因组中通过PCR扩增得到调控因子PadhE-1的基因,并克隆到表达载体pET-28a得到重组质粒pET-PadhE。转化大肠杆菌JM109(DE3),经IPTG诱导表达,Ni2+亲和柱纯化,得到重组表达的调控因子PadhE-1。体外凝胶阻滞实验证实PadhE-1能与TRRadhE特异性结合。这将有助于我们了解嗜热厌氧乙醇杆菌的乙醇代谢调控机理。Thermoanaerobacter ethanolicus produces ethanol from biomass derived substrates at temperatures above 70°C.AdhE,which exhibited bifunctional acetaldehyde/ethanol dehydrogenase activities,play a key role in producing ethanol.Proteins interacting with the transcriptional regulatory region （TRR） of adhE were for the first time isolated and identified by affinity chromatography.One of the proteins,PadhE-1,a homolog of LacI family,was cloned and expressed in Escherichia coli.Through electromobility shift assays,it was determined that the interaction between PadhE-1 and TRRadhE was specific.The research would be helpful to construct the bacteria with the extremely thermophilic and high ethanol-producing characteristics.

关 键 词：嗜热乙醇杆菌JW200 亲和层析 调控因子 凝胶阻滞 

分 类 号：Q78[生物学—分子生物学]