Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation.
Modifications of the E.coli Lac repressor for expression in eukaryotic cells: effects of nuclear signal sequences on protein activity and nuclear accumulation.
复制标题
大肠杆菌 Lac 阻遏物在真核细胞中表达的修饰：核信号序列对蛋白质活性和核积累的影响。
DOI：
10.1093/nar/20.7.1785
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发表时间：
1992
影响因子：
14.9
通讯作者：
Short,JM
中科院分区：
文献类型：
--
作者：
Fieck,A;Wyborski,DL;Short,JM
设计真核表达载体以产生E.构建了靶向哺乳动物细胞核的collLac阻遏蛋白。这些构建体携带在不同位置与来自SV 40大T抗原或腺病毒E1 a的核定位序列（NLS）融合的lac阻遏物基因（lacl）。当NLS与lad基因在5'端融合时，产生的蛋白质比未修饰的LacI蛋白质表现出更紧密的β-半乳糖苷酶表达抑制。基因3'末端的定位序列通常减少IPTG的诱导，而在3'末端上游9个碱基对的SV 40 NLS的引入消除了阻遏物活性。当任一NLS置于随机9个碱基对接头后面的3'末端时，Lacl蛋白的活性取决于接头的序列，并且在测试的10个接头中的9个中，蛋白的活性受到不利影响。一个例外是来自p3'ss的融合蛋白，其在9个碱基对接头AGC AGC CTG（ser-ser-leu）后面的lacl的3'末端具有NLS。该蛋白具有高效的核积累、强的阻遏活性和对IPTG诱导的更高敏感性。来自p3 ′ ss融合蛋白的功能性接头延伸位于蛋白C-末端的亮氨酸拉链七肽重复。这些数据支持亮氨酸拉链在四聚体形成中的作用，并预测该拉链的延伸将进一步稳定蛋白质。这种修饰的lacl基因对于改善原核调节系统对真核细胞的适应性是有价值的。
Eukaryotic expression vectors designed to produceE. collLac repressor protein targeted to the nucleus of mammalian cells were constructed. These constructions carry the lac repressor gene (lacl) fused at different positions to a nuclear localization sequence (NLS) from either the SV40 large T antigen or the adenovirus E1a. When the NLS's were fused to the lad gene at the 5' end, the protein produced exhibited tighter repression of beta-galactosidase expression than the unmodified Lacl protein. Localization sequences at the extreme 3' end of the gene generally diminished induction by IPTG, while Introduction of the SV40 NLS nine base pairs upstream of the 3' end eliminated repressor activity. When either NLS was placed at the 3' end behind a random nine base pair linker, the activity of the Lacl protein depended on the sequence of the linker, and in 9 of 10 linkers tested, activity of the protein was adversely affected. The one exception was the fusion protein from p3'ss, which had the NLS at the 3' end of lacl behind the nine base pair linker, AGC AGC CTG (ser-ser-leu). This protein exhibited efficient nuclear accumulation, strong repressor activity and greater sensitivity to IPTG induction. The functional linker from the p3'ss fusion protein extends the leucine zipper heptad repeat located at the C-termlnus of the protein. These data support the role of the leucine zipper in tetramer formation and predict that extension of this zipper will further stabilize the protein. This modifiedlaclgene should be valuable for improved adaptation of the prokaryotic regulatory system to eukaryotic cells.