用于固态核磁共振的均匀同位素标记KcsA的制备：表达、纯化、重组成脂质体及功能测定。
Preparation of uniformly isotope labeled KcsA for solid state NMR: expression, purification, reconstitution into liposomes and functional assay.
作者信息
Bhate Manasi P, Wylie Benjamin J, Thompson Ameer, Tian Lin, Nimigean Crina, McDermott Ann E
机构信息
Department of Chemistry, Columbia University, NY 10027, United States.
出版信息
Protein Expr Purif. 2013 Oct;91(2):119-24. doi: 10.1016/j.pep.2013.07.013. Epub 2013 Aug 1.
We report the expression, purification, liposome reconstitution and functional validation of uniformly (13)C and (15)N isotope labeled KcsA, a bacterial potassium channel that has high homology with mammalian channels, for solid-state NMR studies. The expression and purification is optimized for an average yield of ∼35-40mg/L of M9 media in a time-efficient way. The protein purity is confirmed by gel electrophoresis and the protein concentration is quantified by UV-vis absorption spectroscopy. Protocols to efficiently reconstitute KcsA into liposomes are also presented. The presence of liposomes is confirmed by cryo-electron microscopy images and the effect of magic angle spinning on liposome packing is shown. High-resolution solid-state NMR spectra of uniformly isotope labeled KcsA in these liposomes reveal that our protocol yields to a very homogenous KcsA sample with high signal to noise and several well-resolved residues in NMR spectra. Electrophysiology of our samples before and after solid-state NMR show that channel function and selectivity remain intact after the solid-state NMR.
我们报告了用于固态核磁共振研究的均匀（13）C和（15）N同位素标记的KcsA（一种与哺乳动物通道具有高度同源性的细菌钾通道）的表达、纯化、脂质体重组和功能验证。以高效的方式对表达和纯化进行了优化，以使M9培养基的平均产量达到约35 - 40mg/L。通过凝胶电泳确认蛋白质纯度，并通过紫外可见吸收光谱法定量蛋白质浓度。还介绍了将KcsA有效重组到脂质体中的方案。通过冷冻电子显微镜图像确认脂质体的存在，并展示了魔角旋转对脂质体堆积的影响。这些脂质体中均匀同位素标记的KcsA的高分辨率固态核磁共振谱表明，我们的方案产生了非常均匀的KcsA样品，具有高信噪比，并且在核磁共振谱中有几个分辨率良好的残基。固态核磁共振前后我们样品的电生理学表明，固态核磁共振后通道功能和选择性保持完整。
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