Taghavian Omid, Mandal Manoj K, Steinmetz Nicole F, Rasche Stefan, Spiegel Holger, Fischer Rainer, Schillberg Stefan
Fraunhofer IME, Forckenbeckstraβe 6, 52074, Aachen, Germany.
Department of Biomedical Engineering, School of Medicine, Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106, USA.
RSC Adv. 2012 Mar 7;2(5):1970-1978. doi: 10.1039/C2RA00857B. Epub 2012 Jan 9.
We describe a novel nanobiotechnology platform based on subviral particles derived from (IBD-SVPs). The major virus coat protein VP2 assembles into spherical, 23 nm SVPs when expressed as a heterologous protein in the yeast . We recovered up to 38 mg of IBD-SVPs at > 95% purity from 1 L of recombinant yeast culture. The purified particles were able to tolerate organic solvents up to 20% concentration (ethanol or dimethylsulfoxide), they resisted temperatures up to 65 °C and remained stable over a wide pH range (2.5-9.0). We achieved bioconjugation to the amine groups of lysine residues and to the carboxyl groups of aspartic and glutamic acid residues, allowing the functionalization of IBD-SVPs with biotin. The accessibility of surface amine groups was measured using Alexa Fluor 488 -hydroxysuccinimide (NHS) ester, an amine-selective fluorescent dye, revealing that approximately 60 dye molecules were attached to the surface of each particle. IBD-SVPs can therefore be exploited as a robust and versatile nanoscaffold to display diverse functional ligands.
我们描述了一种基于源自传染性法氏囊病病毒的亚病毒颗粒(IBD - SVPs)的新型纳米生物技术平台。主要病毒衣壳蛋白VP2在酵母中作为异源蛋白表达时会组装成23纳米的球形SVP。我们从1升重组酵母培养物中回收了高达38毫克、纯度大于95%的IBD - SVPs。纯化后的颗粒能够耐受浓度高达20%的有机溶剂(乙醇或二甲基亚砜),能抵抗高达65°C的温度,并且在较宽的pH范围(2.5 - 9.0)内保持稳定。我们实现了与赖氨酸残基的胺基以及天冬氨酸和谷氨酸残基的羧基的生物共轭,从而使IBD - SVPs能用生物素进行功能化修饰。使用胺选择性荧光染料Alexa Fluor 488 - 羟基琥珀酰亚胺(NHS)酯测量表面胺基的可及性,结果显示每个颗粒表面约附着60个染料分子。因此,IBD - SVPs可被用作一种强大且通用的纳米支架来展示多种功能性配体。