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首页> 外文期刊>Protein Expression and Purification >Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli
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Construction of a modified vector for efficient purification of recombinant Mycobacterium tuberculosis proteins expressed in Escherichia coli

机译：用于有效纯化在大肠杆菌中表达的重组结核分枝杆菌蛋白的修饰载体的构建

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A major problem in assessing the vaccine and diagnostic potential of various proteins encoded by Mycobacterium tuberculosis genome is the inability to produce large quantities of these proteins, even when Escherichia coli or other heterologous systems are employed for recombinant protein production. To overcome these barriers, we have constructed a modified expression vector, using pGEX-4T-1 vector as the backbone. In addition to the features offered by the pGEX-4T vectors, the new vector allowed easy purification of recombinant proteins on the highly versatile Ni-NTA-agarose affinity matrix. The utility of the new vector was demonstrated by expressing and purifying, to near homogeneity, two M. tuberculosis proteins, i.e., Rv3872 (a member of the multigene PE subfamily) and Rv3873 (a member of the multi-gene PPE subfamily), which are encoded by the RD1 region of M. tuberculosis. The proteins encoded by rv3872 and rv3873 were expressed at high levels as fusion proteins with glutathione-S-transferase in E. coli. The recombinant Rv3872 and Rv3873 proteins were purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose and/or Ni-NTA-agarose affinity matrix and cleavage of the purified fusion proteins by thrombin protease. The recombinant Rv3872 protein was nearly homogeneous (more than 95% pure) while Rv3873 preparation was more than 90% pure. The recombinant Rv3872 and Rv3873 proteins were immunologically active and reacted with antibodies in sera from TB patients. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags for efficient expression and purification of recombinant M. tuberculosis proteins expressed in E. coli, which could be used for further diagnostic and immunological studies. (C) 2003 Elsevier Science (USA). All rights reserved. [References: 38]
机译：评估结核分枝杆菌基因组编码的各种蛋白质的疫苗和诊断潜力的主要问题是，即使将大肠杆菌或其他异源系统用于重组蛋白质生产，也无法产生大量这些蛋白质。为了克服这些障碍，我们以pGEX-4T-1载体为骨架构建了修饰的表达载体。除了pGEX-4T载体提供的功能外，新载体还允许在高度通用的Ni-NTA-琼脂糖亲和基质上轻松纯化重组蛋白。通过表达和纯化两个结核分枝杆菌蛋白，即Rv3872（多基因PE亚家族的成员）和Rv3873（多基因PPE亚家族的成员），将新载体的效用得以证明。由结核分枝杆菌的RD1区编码。由rv3872和rv3873编码的蛋白在大肠杆菌中以高水平表达为具有谷胱甘肽S-转移酶的融合蛋白。通过在谷胱甘肽-琼脂糖和/或Ni-NTA-琼脂糖亲和基质上的亲和纯化并通过凝血酶蛋白酶切割纯化的融合蛋白，纯化并分离出重组Rv3872和Rv3873蛋白，使其不含融合伴侣（GST）。重组Rv3872蛋白几乎是均质的（纯度超过95％），而Rv3873制剂的纯度则超过90％。重组Rv3872和Rv3873蛋白具有免疫活性，并与TB患者血清中的抗体反应。我们的结果证明了具有两个亲和标签的新构建的表达载体在大肠杆菌中表达的重组结核分枝杆菌蛋白的有效表达和纯化中的实用性，可用于进一步的诊断和免疫学研究。 （C）2003 Elsevier Science（美国）。版权所有。 [参考：38]

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