Large-scale functional purification of recombinant HIV-1 capsid.
Large-scale functional purification of recombinant HIV-1 capsid.
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DOI：
10.1371/journal.pone.0058035
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Sakowicz R
中科院分区：
文献类型：
--
作者：
Hung M;Niedziela-Majka A;Jin D;Wong M;Leavitt S;Brendza KM;Liu X;Sakowicz R
在人类免疫缺陷病毒1型（HIV-1）病毒粒子成熟过程中，衣壳蛋白发生重大重排，形成保护病毒核蛋白复合物的锥形核心。改变衣壳核心稳定性的衣壳序列突变对病毒的感染性和复制有害。最近，衣壳组装已成为开发新一代抗逆转录病毒药物的有吸引力的靶点。药物筛选工作和随后的结构和机理研究需要克量的活性、均质和纯蛋白质。大肠杆菌表达的重组衣壳蛋白的实验室纯化的常规手段依赖于柱层析步骤，其不适于大规模生产。在这里，我们提出了一种基于功能的纯化野生型和四重突变体衣壳蛋白，这依赖于衣壳蛋白的固有倾向，以纯化和去纯化。该方法不需要填充相当大的色谱柱，并且可以产生两位数克量的功能和生物化学性能良好的蛋白质，纯度大于98%。我们已经使用纯化的衣壳蛋白在我们内部开发的聚合测定中表征两种已知的组装抑制剂，并测量它们的结合亲和力。我们的衣壳纯化程序提供了一种用于纯化HIV-1生命周期中大量关键蛋白的稳健方法，有助于鉴定下一代抗HIV药物。
During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents.
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