Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies
Rapid, high-yield expression and purification of Ca2+-ATPase regulatory proteins for high-resolution structural studies
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DOI：
10.1016/j.pep.2004.11.015
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发表时间：
2005-03-01
影响因子：
1.6
通讯作者：
Young, HS
中科院分区：
文献类型：
--
作者：
Douglas, JL;Trieber, CA;Young, HS
磷蛋白(PLB)和肌磷脂(SLN)是分别调节心肌和骨骼肌钙-ATPase并直接改变其钙转运特性的小分子膜蛋白。PLB在低于最大钙浓度时与心脏的钙-ATPase相互作用并对其进行调节，从而减慢心脏的舒张率，降低心脏的收缩能力。SLN与骨骼肌钙-ATPase相互作用和调节的机制类似于原球蛋白使用的机制。虽然这些调控相互作用在生化和生理上都有很好的特征，但缺乏结构细节。为了进行结构研究，如电子冷冻显微镜和X射线结晶学，需要大量的过度表达和纯化的蛋白质。在此，我们报道了一种快速高效地大量生产PLB和SLN的改进方法。总之，重组野生型PLB和SLN在大肠杆菌中以麦芽糖结合蛋白的形式大量表达。烟草蚀刻病毒的酶切位点允许融合蛋白的特异性切割和重组PLB或SLN的释放。用盐酸胍选择性增溶，再用反相高效液相色谱，可以快速、大规模地生产高纯度蛋白质。重组和ATPase活性的测定证实了我们的重组调控蛋白与钙-ATPase之间的功能相互作用。过量生产的蛋白质的抑制特性与以前的研究一致，后者的抑制作用可以通过提高钙浓度来解除。此外，我们还证明了我们的重组PLB和SLN适用于高分辨结构研究。(C)2004 Elsevier Inc.保留所有权利。
Phospholamban (PLB) and sarcolipin (SLN) are small integral membrane proteins that regulate the Ca2+-ATPases of cardiac and skeletal muscle, respectively, and directly alter their calcium transport properties. PLB interacts with and regulates the cardiac Ca2+-ATPase at submaximal calcium concentrations, thereby slowing relaxation rates and reducing contractility in the heart. SLN interacts with and regulates the skeletal muscle Ca2+-ATPase in a mechanism analogous to that used by PLB. While these regulatory interactions are biochemically and physiologically well characterized, structural details are lacking. To pursue structural studies, such as electron cryo-microscopy and X-ray crystallography, large quantities of over-expressed and purified protein are required. Herein, we report a modified method for producing large quantities of PLB and SLN in a rapid and efficient manner. Briefly, recombinant wild-type PLB and SLN were over-produced in Escherichia coli as maltose binding protein fusion proteins. A tobacco etch virus protease site allowed specific cleavage of the fusion protein and release of recombinant PLB or SLN. Selective solubilization with guanidine-hydrochloride followed by reverse-phase HPLC permitted the rapid, large-scale production of highly pure protein. Reconstitution and measurement of ATPase activity confirmed the functional interaction between our recombinant regulatory proteins and Ca2+-ATPase. The inhibitory properties of the over-produced proteins were consistent with previous studies, where the inhibition was relieved by elevated calcium concentrations. In addition, we show that our recombinant PLB and SLN are suitable for high-resolution structural studies. (c) 2004 Elsevier Inc. All rights reserved.