Prs (phosphoribosyl pyrophosphate synthase) is a broadly conserved protein that synthesises 5-phosphoribosyl 1-pyrophospate (PRPP); a substrate for biosynthesis of at least 10 enzymatic pathways including biosynthesis of DNA building blocks - purines and pyrimidines. In Escherichia coli, it is a protein of homo-hexameric quaternary structure, which can be challenging to work with, due to frequent aggregation and activity loss. Several studies showed brief purification protocols for various bacterial PRPP synthases, in most cases involving ammonium sulfate precipitation. Here, we provide a protocol for expression of E. coli Prs protein in Rosetta (DE3) and BL21 (DE3) pLysE strains and a detailed method for His-Prs and untagged Prs purification on nickel affinity chromatography columns. This protocol allows purification of proteins with high yield, purity and activity. We report here N-terminally His-tagged protein fusions, stable and active, providing that the temperature around 20 °C is maintained at all stages, including centrifugation. Moreover, we successfully applied this method to purify two enzyme variants with K194A and G9S alterations. The K194A mutation in conserved lysine residue results in protein variant unable to synthetize PRPP, while the G9S alteration originates from prs-2 allele variant which was previously related to thermo-sensitive growth. His-PrsG9S protein purified here, exhibited comparable activity as previously observed in-vivo suggesting the proteins purified with our protocol resemble their physiological state. The protocol for Prs purification showed here indicates guidance to improve stability and quality of the protein and to ensure more reliable results in further assays in-vitro.

中文翻译：

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高质量的带有His标签和无标签的大肠杆菌磷酸核糖磷酸合酶（Prs）纯化的可靠方法。

Prs（磷酸核糖基焦磷酸合酶）是一种广泛保存的蛋白质，可合成5-磷酸核糖基1-焦磷酸酯（PRPP）；生物合成至少10种酶促途径的底物，包括生物合成DNA构件-嘌呤和嘧啶。在大肠杆菌中，它是一种具有同六聚体的四级结构的蛋白质，由于频繁的聚集和活性丧失，很难与之竞争。多项研究表明，各种细菌PRPP合成酶的简要纯化方案，大多数情况下涉及硫酸铵沉淀。在这里，我们提供了在Rosetta（DE3）和BL21（DE3）pLysE菌株中表达大肠杆菌Prs蛋白的方案，以及在镍亲和色谱柱上进行His-Prs和未标记Prs纯化的详细方法。该方案可高纯度纯化蛋白质，纯度和活​​性。我们在这里报告N末端带有His标记的蛋白融合物，稳定且活跃，只要在包括离心作用在内的所有阶段都保持20°C左右的温度即可。此外，我们成功地将该方法用于纯化具有K194A和G9S改变的两种酶变体。保守的赖氨酸残基中的K194A突变导致蛋白质变异体无法合成PRPP，而G9S变异源自prs-2等位基因变异体，该变异体先前与热敏性生长有关。在此纯化的His-PrsG9S蛋白显示出与以前体内观察到的活性相当的活性，表明用我们的方案纯化的蛋白类似于它们的生理状态。