Araujo Mariana S, Baura Valter A, Souza Emanuel M, Benelli Elaine M, Rigo Liu U, Steffens M Berenice R, Pedrosa Fabio O, Chubatsu Leda S
Department of Biochemistry and Molecular Biology, Universidade Federal do Paraná, CP19046, Curitiba, PR 81531-990, Brazil.
Protein Expr Purif. 2004 Jan;33(1):19-24. doi: 10.1016/j.pep.2003.08.024.
Azospirillum brasilense is a diazotroph which associates with important agricultural crops. The nitrogen fixation process in this organism is highly regulated by ammonium and oxygen, and involves several proteins including the two PII-like proteins, GlnB and GlnZ. Although these proteins are structurally very similar, they play different roles in the control of nitrogen fixation. In this work, we describe the expression, purification, and uridylylation of the GlnZ protein of A. brasilense strain FP2. The amplified glnZ gene was sub-cloned and expressed as a His-tagged fusion protein. After purification, we obtained 30-40 mg of purified GlnZ per liter of culture. This protein was purified to 99% purity and assayed for in vitro uridylylation using a partially purified Escherichia coli GlnD as a source of uridylylyl-transferase activity. Analyses of the uridylylation reactions in non-denaturing and denaturing polyacrylamide gel electrophoresis showed that up to 74% of GlnZ monomers were modified after 30 min reaction. This covalent modification is strictly dependent on ATP and 2-ketoglutarate, while glutamine acts as an inhibitor and promotes deuridylylation.
巴西固氮螺菌是一种与重要农作物共生的固氮菌。该生物体中的固氮过程受到铵和氧的高度调控，涉及多种蛋白质，包括两种类PII蛋白GlnB和GlnZ。尽管这些蛋白质在结构上非常相似，但它们在固氮控制中发挥着不同的作用。在这项工作中，我们描述了巴西固氮螺菌FP2菌株GlnZ蛋白的表达、纯化和尿苷酸化。扩增的glnZ基因被亚克隆并表达为His标签融合蛋白。纯化后，每升培养物可获得30 - 40毫克纯化的GlnZ。该蛋白被纯化至99%的纯度，并使用部分纯化的大肠杆菌GlnD作为尿苷酰转移酶活性来源进行体外尿苷酸化测定。在非变性和变性聚丙烯酰胺凝胶电泳中对尿苷酸化反应的分析表明，反应30分钟后，高达74%的GlnZ单体被修饰。这种共价修饰严格依赖于ATP和2 - 酮戊二酸，而谷氨酰胺起抑制剂作用并促进去尿苷酸化。
