OVERPRODUCTION AND PURIFICATION OF THE LUXR GENE-PRODUCT - TRANSCRIPTIONAL ACTIVATOR OF THE VIBRIO-FISCHERI LUMINESCENCE SYSTEM
OVERPRODUCTION AND PURIFICATION OF THE LUXR GENE-PRODUCT - TRANSCRIPTIONAL ACTIVATOR OF THE VIBRIO-FISCHERI LUMINESCENCE SYSTEM
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DOI：
10.1073/pnas.84.19.6639
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发表时间：
1987-10-01
影响因子：
11.1
通讯作者：
GREENBERG, EP
中科院分区：
文献类型：
--
作者：
KAPLAN, HB;GREENBERG, EP
费氏弧菌发光基因的表达需要一种诱导物（称为自诱导物）和一种正调控元件（luxR基因产物）。将含有在tac启动子控制下的luxR的质粒工程化以过量产生该基因产物。过量产生的luxR基因产物在体内是有活性的，并且其表观单体分子量与由luxR在其自身启动子控制下编码的蛋白质（Mr 27，000）是不可区分的。新的tac-luxR构建体指导在缺乏其他lux基因的诱导大肠杆菌细胞中合成大量的luxR基因产物。在其它lux基因存在下，未检测到tac-luxR构建体的过表达。过量产生的luxR基因产物，形成细胞质包涵体，纯化并用于后续研究。非平衡pH梯度电泳表明该蛋白为碱性蛋白，并测定了其氨基端15个氨基酸的序列。通过膜过滤结合试验检测DNA结合活性;在所用条件下，结合不是lux DNA特异性的。未检测到氚标记的自诱导物与luxR基因产物的结合，并且自诱导物增强luxR基因产物与DNA的结合不能重复检测。
Expression of Vibrio fischeri luminescence genes requires an inducer, termed autoinducer, and a positive regulatory element, the luxR gene product. A plasmid containing luxR under control of a tac promoter was engineered to overproduce this gene product. The overproduced luxR gene product was active in vivo, and its apparent monomeric molecular weight was indistinguishable from that of the protein encoded by luxR under control of its own promoter (Mr 27,000). The new tac-luxR construct directed the synthesis of large quantities of the luxR gene product in induced Escherichia coli cells lacking other lux genes. In the presence of the other lux genes, overexpression of the tac-luxR construct was not detected. The overproduced luxR gene product, which formed cytoplasmic inclusion bodies, was purified and used in subsequent studies. Nonequilibrium pH gradient electrophoresis indicated that the protein was basic, and the amino-terminal 15 amino acids were sequenced. DNA-binding activity was detected by membrane filter binding assay; under the conditions used, the binding was not lux DNA-specific. Binding of tritium-labeled autoinducer to the luxR gene product was not detected, and autoinducer enhancement of the binding of the luxR gene product to DNA could not be detected reproducibly.