Kulkarni Amod P, Kellaway Lauriston A, Kotwal Girish J
Division of Anatomical Pathology, Department of Clinical Laboratory sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
Methods Mol Biol. 2012;890:289-303. doi: 10.1007/978-1-61779-876-4_17.
Poxviruses are one of the most complex of animal viruses and encode for over 150 proteins. The interactions of many of the poxviral-encoded proteins with host proteins, as well as with other proteins, such as transcription complexes, have been well characterized at the qualitative level. Some have also been characterized quantitatively by two hybrid systems and surface plasmon resonance approaches. Presented here is an alternative approach that can enable the understanding of complex interactions with multiple ligands. The example given is that of vaccinia virus complement control protein (VCP). The complement system forms the first line of defense against microorganisms and a failure to appropriately regulate it is implicated in many inflammatory disorders, such as traumatic brain injury, Alzheimer's disease (AD), and rheumatoid arthritis. The complement component C3 is central to the complement activation. Complement regulatory proteins, capable of binding to the central complement component C3, may therefore effectively be employed for the treatment and prevention of these disorders. There are many biochemical and/or immunoassays available to study the interaction of proteins with complement components. However, protocols for many of them are time consuming, and not all assays are useful for multiple screening. In addition, most of these assays may not give information regarding the nature of binding, the number of molecules interacting with the complement component C3, as well as kinetics of binding. Some of the assays may require labeling which may induce changes in protein confirmation. We report a protocol for an assay based on quartz crystal microbalance with dissipation monitoring (QCM-D) technology, which can effectively be employed to study poxviral proteins for their ability to interact with their ligand. A protocol was developed in our laboratories to study the interaction of VCP with the complement component C3 using Q-sense (D-300), equipment based on QCM-D technology. The protocol can also be used as a prototype for studying both proteins and small-sized compounds (for use as anti-poxvirals) for their ability to interact with and/or inhibit the activity of their ligands.
痘病毒是最复杂的动物病毒之一，可编码150多种蛋白质。许多痘病毒编码的蛋白质与宿主蛋白质以及其他蛋白质（如转录复合物）之间的相互作用，在定性水平上已得到充分表征。其中一些还通过双杂交系统和表面等离子体共振方法进行了定量表征。本文介绍了一种替代方法，该方法能够理解与多种配体的复杂相互作用。给出的例子是牛痘病毒补体控制蛋白（VCP）。补体系统构成了抵御微生物的第一道防线，未能对其进行适当调节与许多炎症性疾病有关，如创伤性脑损伤、阿尔茨海默病（AD）和类风湿性关节炎。补体成分C3是补体激活的核心。因此，能够结合补体核心成分C3的补体调节蛋白可有效地用于治疗和预防这些疾病。有许多生化和/或免疫测定方法可用于研究蛋白质与补体成分之间的相互作用。然而，其中许多方法的操作流程耗时较长，并非所有测定方法都适用于多重筛选。此外，这些测定方法大多可能无法提供有关结合性质、与补体成分C3相互作用的分子数量以及结合动力学的信息。有些测定方法可能需要标记，这可能会导致蛋白质构象发生变化。我们报告了一种基于石英晶体微天平耗散监测（QCM-D）技术的测定方法，该方法可有效地用于研究痘病毒蛋白与配体相互作用的能力。我们实验室开发了一种使用基于QCM-D技术的Q-sense（D-300）设备研究VCP与补体成分C3相互作用的方法。该方法还可作为研究蛋白质和小型化合物（用作抗痘病毒药物）与配体相互作用和/或抑制其活性能力的原型。
