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Engineering Potato Virus X Particles for a Covalent Protein Based Attachment of Enzymes
Small ( IF 13.0 ) Pub Date : 2017-11-10 , DOI: 10.1002/smll.201702151 Juliane Röder 1 , Rainer Fischer 1 , Ulrich Commandeur 1
Small ( IF 13.0 ) Pub Date : 2017-11-10 , DOI: 10.1002/smll.201702151 Juliane Röder 1 , Rainer Fischer 1 , Ulrich Commandeur 1
Affiliation
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Plant virus nanoparticles are often used to display functional amino acids or small peptides, thus serving as building blocks in application areas as diverse as nanoelectronics, bioimaging, vaccination, drug delivery, and bone differentiation. This is most easily achieved by expressing coat protein fusions, but the assembly of the corresponding virus particles can be hampered by factors such as the fusion protein size, amino acid composition, and post‐translational modifications. Size constraints can be overcome by using the Foot and mouth disease virus 2A sequence, but the compositional limitations cannot be avoided without the introduction of time‐consuming chemical modifications. SpyTag/SpyCatcher technology is used in the present study to covalently attach the Trichoderma reesei endoglucanase Cel12A to Potato virus X (PVX) nanoparticles. The formation of PVX particles is confirmed by western blot, and the ability of the particles to display Cel12A is demonstrated by enzyme‐linked immunosorbent assays and transmission electron microscopy. Enzymatic assays show optimal reaction conditions of 50 °C and pH 6.5, and an increased substrate conversion rate compared to free enzymes. It is concluded that PVX displaying the SpyTag can serve as new scaffold for protein display, most notably for proteins with post‐translational modifications.
中文翻译：
工程马铃薯病毒X颗粒，用于基于共价蛋白的酶附着
植物病毒纳米粒子通常用于展示功能性氨基酸或小肽，因此在诸如纳米电子学，生物成像，疫苗接种，药物递送和骨骼分化等各种应用领域中充当构建模块。通过表达外壳蛋白融合蛋白最容易实现，但是相应的病毒颗粒的组装可能会受到诸如融合蛋白大小，氨基酸组成和翻译后修饰等因素的阻碍。通过使用口蹄疫病毒2A序列可以克服大小限制，但是如果不引入费时的化学修饰，就无法避免成分上的限制。SpyTag / SpyCatcher技术在本研究中用于共价附着里氏木霉内切葡聚糖酶Cel12A转化为马铃薯X病毒（PVX）纳米颗粒。通过蛋白质印迹证实了PVX颗粒的形成，并且通过酶联免疫吸附测定和透射电子显微镜证明了颗粒展示Cel12A的能力。酶法测定显示最佳反应条件为50°C和pH 6.5，与游离酶相比，底物转化率提高。结论是，展示SpyTag的PVX可以作为蛋白质展示的新支架，最显着的是具有翻译后修饰的蛋白质。
更新日期：2017-11-10
中文翻译：
工程马铃薯病毒X颗粒，用于基于共价蛋白的酶附着
植物病毒纳米粒子通常用于展示功能性氨基酸或小肽，因此在诸如纳米电子学，生物成像，疫苗接种，药物递送和骨骼分化等各种应用领域中充当构建模块。通过表达外壳蛋白融合蛋白最容易实现，但是相应的病毒颗粒的组装可能会受到诸如融合蛋白大小，氨基酸组成和翻译后修饰等因素的阻碍。通过使用口蹄疫病毒2A序列可以克服大小限制，但是如果不引入费时的化学修饰，就无法避免成分上的限制。SpyTag / SpyCatcher技术在本研究中用于共价附着里氏木霉内切葡聚糖酶Cel12A转化为马铃薯X病毒（PVX）纳米颗粒。通过蛋白质印迹证实了PVX颗粒的形成，并且通过酶联免疫吸附测定和透射电子显微镜证明了颗粒展示Cel12A的能力。酶法测定显示最佳反应条件为50°C和pH 6.5，与游离酶相比，底物转化率提高。结论是，展示SpyTag的PVX可以作为蛋白质展示的新支架，最显着的是具有翻译后修饰的蛋白质。
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