Saïda Fakhri, Uzan Marc, Lallemand Jean-Yves, Bontems François
Laboratoire ICSN-RMN, Institut de Chimie des Substances Naturelles, Ecole Polytechnique, Route de Saclay, 91128, Palaiseau Cedex, France,
Biotechnol Prog. 2003 May-Jun;19(3):727-33. doi: 10.1021/bp0257224.
By coupling the toxic restriction endoribonuclease RegB, from the bacteriophage T4, to the prokaryotic T7 and the eukaryotic GAL1 promoters, we constructed a two-function plasmid called pTOXR-1. This plasmid is a zero-background cloning vector. It allows an efficient positive selection of recombinant plasmids without the need to completely digest, dephosphorylate, or purify the vector prior to the ligation step. The pTOXR-1 positive selection system requires no special Escherichia coli strains, no special culture media, and no addition of inducer to the selective plates. In addition, since this vector carries all signals required for both prokaryotic and eukaryotic expression, it allows the overproduction of heterologous proteins, fused to a polyhistidine tag, in the bacterium E. coli and in the yeast Saccharomyces cerevisiae from a single plasmid. Hence, this vector may be a useful time-saving tool for one-step cloning and versatile protein expression in bacteria and yeast.
通过将来自噬菌体T4的有毒限制性核糖核酸内切酶RegB与原核T7启动子和真核GAL1启动子偶联，我们构建了一种名为pTOXR - 1的双功能质粒。该质粒是一种零背景克隆载体。它允许对重组质粒进行高效的阳性筛选，而无需在连接步骤之前对载体进行完全消化、去磷酸化或纯化。pTOXR - 1阳性筛选系统不需要特殊的大肠杆菌菌株、特殊的培养基，也不需要在选择平板中添加诱导剂。此外，由于该载体携带了原核和真核表达所需的所有信号，它允许从单个质粒在大肠杆菌和酿酒酵母中过量表达与多组氨酸标签融合的异源蛋白质。因此，该载体可能是一种用于细菌和酵母中一步克隆和多功能蛋白质表达的有用的节省时间的工具。
