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首页> 外文期刊>Protein Expression and Purification >Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies
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Overexpression, purification, and characterization of recombinant Ca-ATPase regulators for high-resolution solution and solid-state NMR studies

机译：用于高分辨率溶液和固态NMR研究的重组Ca-ATPase调节剂的过表达，纯化和表征

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Phospholamban (PLB) and Sarcolipin (SLN) are integral membrane proteins that regulate muscle contractility via direct interaction with the Ca-ATPase in cardiac and skeletal muscle, respectively. The molecular details of these protein-protein interactions are as vet undetermined. Solution and solid-state NMR spectroscopies have proven to be effective tools for deciphering such regulatory mechanisms to a high degree of resolution; however, large quantities of pure recombinant protein are required for these studies. Thus. recombinant PLB and SLN production in Escherichia coli was optimized for use in NMR experiments. Fusions of PLB and SLN to maltose binding protein (MBP) were constructed and optimal conditions for protein expression and purification were screened. This facilitated the large-scale production of highly pure protein. To confirm their functionality, the biological activities of recombinant PLB and SLN were compared to those of their synthetic counterparts. The regulation of Ca-ATPase activity by recombinant PLB and SLN was indistinguishable from the regulation by synthetic proteins, demonstrating the functional integrity of the recombinant constructs and ensuring the biological relevance of our future structural studies. Finally, NMR spectroscopic conditions were established and optimized for use in investigations of the mechanism of Ca-ATPase regulation by PLB and SLN. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 24]
机译：Phospholamban（PLB）和Sarcolipin（SLN）是不可或缺的膜蛋白，通过分别与心肌和骨骼肌中的Ca-ATPase直接相互作用来调节肌肉的收缩力。这些蛋白质-蛋白质相互作用的分子细节尚未确定。溶液和固态NMR光谱学已被证明是将此类调控机制解读为高解析度的有效工具。但是，这些研究需要大量的纯重组蛋白。从而。优化了大肠杆菌中重组PLB和SLN的生产，以用于NMR实验。构建了PLB和SLN与麦芽糖结合蛋白（MBP）的融合体，并筛选了蛋白质表达和纯化的最佳条件。这促进了高纯度蛋白质的大规模生产。为了确认其功能，将重组PLB和SLN的生物学活性与其合成对应物的生物学活性进行了比较。重组PLB和SLN对Ca-ATPase活性的调节与合成蛋白的调节没有区别，证明了重组构建体的功能完整性，并确保了我们未来结构研究的生物学意义。最后，建立并优化了NMR光谱条件，用于研究PLB和SLN调控Ca-ATPase的机制。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：24]

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