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Improved Strains and Plasmid Vectors for Conditional Overexpression of His-Tagged Proteins in Haloferax volcanii

机译:改良的菌株和质粒载体,用于在Haloferax volcanii中有条件地表达His标记蛋白

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Research into archaea will not achieve its full potential until systems are in place to carry out genetics and biochemistry in the same species. Haloferax volcanii is widely regarded as the best-equipped organism for archaeal genetics, but the development of tools for the expression and purification of H. volcanii proteins has been neglected. We have developed a series of plasmid vectors and host strains for conditional overexpression of halophilic proteins in H. volcanii . The plasmids feature the tryptophan-inducible p. tnaA promoter and a 6×His tag for protein purification by metal affinity chromatography. Purification is facilitated by host strains, where pitA is replaced by the ortholog from Natronomonas pharaonis . The latter lacks the histidine-rich linker region found in H. volcanii PitA and does not copurify with His-tagged recombinant proteins. We also deleted the mrr restriction endonuclease gene, thereby allowing direct transformation without the need to passage DNA through an Escherichia coli dam mutant.
机译:直到建立了在同一物种中进行遗传和生物化学的系统,对古细菌的研究才能充分发挥其潜力。 Haloferax volcanii被广泛认为是古细菌遗传学中装备最完善的有机体,但是人们忽略了表达和纯化H. volcanii蛋白的工具的开发。我们已经开发了一系列的质粒载体和宿主菌株,用于在火山杆菌中有条件地嗜盐蛋白的过表达。质粒具有色氨酸诱导的p。 tnaA启动子和6×His标签,用于通过金属亲和层析纯化蛋白质。宿主菌株促进了纯化,其中pitA被来自法老对虾的直向同源物替代。后者缺乏在火山嗜血杆菌PitA中发现的富含组氨酸的接头区域,并且不与带有His标签的重组蛋白共纯化。我们还删除了mrr限制性核酸内切酶基因,从而允许直接转化而无需使DNA通过大肠杆菌dam突变体。

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