Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments.
Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments.
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来自大肠杆菌的含组氨酸蛋白质的二维 1H NMR 研究。
DOI：
10.1021/bi00371a071
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发表时间：
1986
期刊：
影响因子：
2.9
通讯作者：
Waygood,EB
中科院分区：
文献类型：
--
作者：
Klevit,RE;Drobny,GP;Waygood,EB
材料与方法样品制备.如前所述（Waygood & Sleeves，1980），从大肠杆菌P650中纯化HPr。在纯化的最后一步之后，将蛋白质从H2O中冻干。为了制备用于NMR实验的HPr样品，将蛋白质溶解在4 mL 5 mM磷酸钾缓冲液（pH 6.5）中，并对相同的缓冲液进行彻底透析。再次将样品冻干并溶解在0.4 mL 99.96%D20或90%H2O/10%D20中，离心除去任何不溶物质，并放入5 mm NMR管中。NMR样品中HPr的最终浓度为约5 mM，溶于50 mM磷酸钾缓冲液（pH 6.5）中。
Materials and MethodsSample Preparation. HPr was purified from Escherichia coli P650 as previously described (Waygood & Sleeves, 1980). Following the last step in the purification, the protein was lyophilized from H20. To prepare HPr samples for the NMR experiments, the protein was dissolved in 4 mL of 5mM potassium phosphate buffer, pH 6.5, and dialyzed exhaustively against the same buffer. The sample was again lyophilized and dissolved in 0.4 mL of either 99.96% D20 or 90% H2O/10% D20, centrifuged to remove any insoluble material, and putinto a 5-mm NMR tube. The final concentration of HPr in the NMR sample was~ 5 mM, in 50 mM potassium phosphate buffer, pH 6.5.