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首页> 外文期刊>Protein Expression and Purification >A new protocol for high-yield purification of recombinant human prothymosin alpha expressed in Escherichia coli for NMR studies
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A new protocol for high-yield purification of recombinant human prothymosin alpha expressed in Escherichia coli for NMR studies

机译：高产率纯化在大肠杆菌中表达的重组人胸腺肽α的新方法用于NMR研究

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摘要

Human prothymosin alpha (ProT alpha) is a small acidic protein (12.1 kDa; pI similar to 3.5) ubiquitously expressed in a wide variety of tissues. The amino acid composition of this protein is highly unusual. While close to half of its sequence is composed of acidic amino acids. the protein does not contain any aromatic residues. ProTa has been shown to play crucial roles in different biological processes including cell proliferation, transcriptional regulation and apoptosis. Despite the multiple functions this protein has, it does not adopt a stable tertiary fold under physiological conditions. In order to understand how ProTot functions, detailed structural characterization of this protein is essential. Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for elucidating the protein structure and dynamics at the atomic level. However, milligrams of isotopically labeled protein with high purity are usually required for the studies. In this work, we developed a high-yield protocol for purifying recombinant ProTot expressed in Escherichia coli by exploiting the intrinsically disordered and acidic natures of this protein. By combining the heat-cooling extraction, ammonium sulfate precipitation, and anion exchange chromatography, we were able to obtain over 20 mg of ProTa with > 97% purity from 1 L of M9 minimal media culture. The new purification protocol provides a cost effective and an efficient way to produce large quantities of high purity recombinant human ProTot in various isotopically labeled forms, which will greatly facilitate the structural studies of this protein by NMR and other biophysical methods. (c) 2007 Elsevier Inc. All rights reserved.
机译：人胸腺肽α（ProT alpha）是一种小的酸性蛋白（12.1 kDa; pI类似于3.5），在各种各样的组织中普遍表达。这种蛋白质的氨基酸组成非常不寻常。虽然其序列的近一半由酸性氨基酸组成。该蛋白质不含任何芳香族残基。已证明ProTa在不同的生物学过程中起关键作用，包括细胞增殖，转录调节和凋亡。尽管该蛋白具有多种功能，但在生理条件下它并不具有稳定的三级折叠。为了了解ProTot的功能，此蛋白的详细结构表征至关重要。核磁共振（NMR）光谱技术是一种在原子水平上阐明蛋白质结构和动力学的强大技术。但是，研究通常需要毫克的高纯度同位素标记蛋白质。在这项工作中，我们开发了一种高产方案，通过利用该蛋白的内在无序和酸性特性来纯化在大肠杆菌中表达的重组ProTot。通过将热冷却萃取，硫酸铵沉淀和阴离子交换色谱相结合，我们能够从1 L的M9基本培养基中获得超过20 mg的ProTa，其纯度> 97％。新的纯化方案为生产各种同位素标记形式的大量高纯度重组人ProTot提供了一种经济高效的方法，这将极大地促进通过NMR和其他生物物理方法对该蛋白的结构研究。 （c）2007 Elsevier Inc.保留所有权利。

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