Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography.
Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography.
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通过离子交换色谱法纯化 RanGDP、RanGTP 和 RanGMPPNP。
DOI：
10.1016/j.ab.2004.06.017
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Yaseen,NabeelR
中科院分区：
文献类型：
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作者：
Bibak,Niloufar;Paul,RachelleMJ;Freymann,DouglasM;Yaseen,NabeelR
RAN是一种小的GTP酶，在鸟苷二磷酸(GDP)结合型(RanGDP)和鸟苷三磷酸(GTP)结合型(RanGTP)之间循环，在核运输和有丝分裂中发挥重要作用。对于研究RAN的功能及其与伴侣蛋白的相互作用，纯化的RanGDP和RanGTP复合体是至关重要的。RAN与非水解性GTP类似物GMPPNP(RanGMPPNP)络合，在需要抑制水解时，用RAN代替RanGTP。在这项研究中，我们证明了RAN与UNO Q离子交换柱的结合对氯化镁浓度的微小变化非常敏感，并利用这一性质纯化了重组RanGTP、RanGMPPNP和RanGDP络合物。在10 mM的氯化镁溶液中，RAN主要存在于穿透过程中，因此从绝大多数细菌蛋白中分离出来。在将氯化镁浓度降至5 mM后，通过离子交换柱负载和氯化钠梯度洗脱，进一步纯化了RanGTP、RanGMPPNP和RanGDP。通过释放结合的核苷酸并用高效液相色谱(HPLC)检查其与已知核苷酸的比对来确认所得到的制剂的纯度。为了进一步证实RAN制剂的纯度和功能，进行了适当的蛋白质结合、酶和核进口试验。这些方法应该有助于通过提供纯功能的RAN-核苷酸复合体来研究涉及RAN的细胞过程。
Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl2concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10mM MgCl2, Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl2to 5mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran–nucleotide complexes.