Sequence-specific 1H, 13C and 15N resonance assignments of the C-terminal domain of KaiA, a circadian clock protein.
Sequence-specific 1H, 13C and 15N resonance assignments of the C-terminal domain of KaiA, a circadian clock protein.
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生物钟蛋白 KaiA C 端结构域的序列特异性 1H、13C 和 15N 共振分配。
DOI：
10.1023/b:jnmr.0000015373.13794.c7
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发表时间：
2004
影响因子：
2.7
通讯作者：
LiWang,AndyC
中科院分区：
文献类型：
--
作者：
Vakonakis,Ioannis;LiWang,AndyC
方法与结果：将长毛虫编码180-283位氨基酸残基的基因亚克隆到pET-32a+载体上，转化大肠杆菌BL21(DE3)。细菌的生长温度为37◦C，以15NH4Cl为唯一氮源，以13C6-葡萄糖或未标记葡萄糖为唯一氮源。在IPTG中加入1 mm的细胞培养液诱导细胞，5h后离心收获细胞，再悬浮细胞颗粒，经过两次法国压力机，细胞裂解物在2000g下离心30min。重组蛋白经金属亲和层析纯化后，用肠激酶法切割，在ThkaiA180C的N-端增加了3个氨基酸残基(AMA)。通过第二步金属亲和层析从混合物中分离出硫氧还蛋白，并通过阴离子交换层析进一步纯化ThkaiA180C。用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析蛋白质纯度。对于双标记，通常从1个L培养物中获得14 mg ThkaiA180C。在这里使用的核磁共振条件下，ThKaiA180C在大约24小时内就很容易被氧化，因此我们获得了氧化形式的完整归属。核磁共振波谱：核磁共振样品在50◦C时含有20 mM氯化钠，20 mM磷酸二氢钠在50◦C下(在23 mBC时，pH 7.07)，在95%H2O/5%D2O混合溶剂或100%D2O中，含有0.02%NaN3，0.1mM DSS，1.2 mM氧化ThkaiA180C。这些光谱是在德克萨斯州农工大学生物分子核磁共振实验室以50◦C的瓦里安·伊诺瓦600兆赫和500兆赫光谱仪上记录的。1H、13C及
Methods and resultsProtein expression and purification: The gene coding for residues 180-283 of T. elongatus KaiA was subcloned into pET-32a+ vector and Escherichia coli BL21 (DE3) was transformed with the resulting plasmid. Bacteria were grown at 37◦ C in minimal medium containing 15NH4Cl as the only nitrogen source, and with either 13C6-glucose or unlabeled glucose. Cells were induced by making the cell culture 1 mM in IPTG and harvested by centrifugation after 5 h. The cell pellet was resuspended, passed through a French press twice and cell lysates were centrifuged at 20,000 g for 30 min. The recombinant protein was purified by metal-affinity chromatography and cleaved with enterokinase, which results in the addition of three residues (AMA) to the N-terminus of ThKaiA180C. Thioredoxin was separated from the mixture by a second metal-affinity chromatography step and ThKaiA180C was further purified by anion exchange chromatography. Protein purity was analyzed using SDS-polyacrylamide gel electrophoresis. For double labeling typically 14 mg ThKaiA180C were obtained from 1 L of culture. ThKaiA180C was readily oxidized under the NMR conditions used here in approximately 24 h, therefore we proceeded to obtain complete assignments for the oxidized form. NMR spectroscopy: NMR samples contained 20 mM NaCl, 20 mM sodium phosphate pH 7.0 at 50◦ C (pH 7.07 at 23◦ C), 0.02% NaN3, 0.1 mM DSS, 1.2 mM oxidized ThKaiA180C in a 95% H2O/5% D2O solvent mixture or 100% D2O. The spectra were recorded on Varian Inova 600 MHz and 500 MHz spectrometers at 50◦ C at the Biomolecular NMR Laboratory at Texas A&M University. 1H, 13C and