钝齿棒杆菌ArgR蛋白的表达、纯化及其多聚体的形成  

Expression and Purification of Corynebacterium crenatum ArgR Protein and Its Polymer Formation

作　　者：袁永[1] 焦海涛[2] 徐峰[1] 彭福中[2] 熊勇华[1] 陈雪岚[2] 

机构地区：[1]南昌大学生命科学与食品工程学院,食品科学与技术国家重点实验室,江西南昌330047 [2]江西师范大学生命科学学院,江西南昌330022

出　　处：《食品科学》2011年第17期210-214,共5页Food Science

基　　金：国家自然科学基金项目(30960012)

摘　　要：通过PCR技术从钝齿棒杆菌(Corynebacterium crenatum)AS1.542基因组中扩增得到长度为516bp的argR基因,将其连接到原核表达载体pET22b(+),并转化至大肠杆菌BL21(DE3)获得重组菌株BL21(DE3)/pET22b-argR,以异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在33℃诱导8h实现高效可溶性表达,获得了一个分子质量为20kD的融合蛋白ArgR。用纯化后的目的蛋白免疫小鼠制备多克隆抗体,间接ELISA检测抗体效价高达1:160000。Western blot分析结果表明,L-精氨酸介导重组蛋白形成的多聚体,可与自制的鼠抗ArgR多克隆抗体进行特异性反应,与预期结果一致,表明ArgR通过与精氨酸共孵育形成了多聚体,该结果有助于采用凝胶阻滞方法对ArgR蛋白与钝齿棒杆菌精氨酸生物合成途径中各操作子的结合情况进行进一步研究。Gene fragment argR from Corynebacterium crenatum was amplified by PCR and inserted into pET22b（＋） vector to construct a recombinant plasmid pET22b-argR.The recombinant plasmid pET22b-argR was transformed into E.coli BL21（DE3） for protein expression.The protein ArgR with molecular weight of 20 kD was successfully expressed in E.coli BL21（DE3）under the induction of IPTG at 33 ℃ for 8 h.The ArgR fusion protein was purified by Ni2＋ affinity column chromatography for preparing polyclonal antibody.ELISA analysis showed that the antibody titer was 1：160000.Western blot analysis revealed that the antibody could react specifically with the expressed recombinant protein and that the purified ArgR was stable in the form of polymer mediated by L-arginine.These investigations can provide a theoretical reference for detecting the interaction between ArgR and promoters from arginine biosynthetic pathway of C.crenatum.

关 键 词：钝齿棒杆菌 argR基因 高效表达 亲和纯化 多聚体 

分 类 号：Q819[生物学—生物工程]