Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax Duffy-binding protein
Biochemical, biophysical, and functional characterization of bacterially expressed and refolded receptor binding domain of Plasmodium vivax Duffy-binding protein
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DOI：
10.1074/jbc.m101531200
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发表时间：
2001-05-18
影响因子：
4.8
通讯作者：
Chitnis, C
中科院分区：
文献类型：
--
作者：
Singh, S;Pandey, K;Chitnis, C
疟原虫对红细胞的侵入是由特异性分子相互作用介导的。间日疟原虫完全依赖于与Duffy血型抗原的相互作用来侵入人红细胞。在侵入期间结合Duffy抗原的间日疟原虫Duffy结合蛋白属于红细胞结合蛋白家族，其还包括恶性疟原虫唾液酸结合蛋白和诺氏疟原虫Duffy结合蛋白。这些蛋白的受体结合结构域位于保守的N-末端，富含半胱氨酸的区域，区域II，在这些蛋白质中发现。在此，我们在大肠杆菌中表达了间日疟原虫Duffy结合结构域PvRII，重组PvRII错误折叠并积累在包涵体中。我们已经开发了方法来重折叠和纯化重组PvRII在其功能构象。生物化学、生物物理和功能表征证实重组PvRII是纯的、均质的和功能活性的，因为其特异性结合Huffy阳性人红细胞。重折叠的PvRII是高免疫原性和高效价的抗体，其可以抑制间日疟原虫Huffy结合蛋白与红细胞的结合，为其作为间日疟原虫疟疾的候选疫苗的开发提供支持。开发生产功能活性重组PvRII的方法是结构研究以及疫苗开发的重要步骤。
Invasion of erythrocytes by malaria parasites is mediated by specific molecular interactions. Plasmodium vivax is completely dependent on interaction with the Duffy blood group antigen to invade human erythrocytes, The P, vivax Duffy-binding protein, which binds the Duffy antigen during invasion, belongs to a family of erythrocyte-binding proteins that also includes Plasmodium falciparum sialic acid binding protein and Plasmodium knowlesi Duffy binding protein, The receptor binding domains of these proteins lie in a conserved, N-terminal, cysteine-rich region, region II, found in each of these proteins. Here, we have expressed P. vivax region II (PvRII), the P. vivax Duffy binding domain, in Escherichia coli, Recombinant PvRII is incorrectly folded and accumulates in inclusion bodies. We have developed methods to refold and purify recombinant PvRII in its functional conformation. Biochemical, biophysical, and functional characterization confirms that recombinant PvRII is pure, homogeneous, and functionally active in that it binds Huffy-positive human erythrocytes with specificity. Refolded PvRII is highly immunogenic and elicits high titer antibodies that can inhibit binding of P. vivax Huffy-binding protein to erythrocytes, providing support for its development as a vaccine candidate for P. vivax malaria. Development of methods to produce functionally active recombinant PvRII is an important step for structural studies as well as vaccine development.