The structural orientation of antibody layers bound to engineered biosensor surfaces
The structural orientation of antibody layers bound to engineered biosensor surfaces
复制标题
DOI：
10.1016/j.biomaterials.2011.01.026
复制
发表时间：
2011-04-01
期刊：
影响因子：
14
通讯作者：
Lakey, Jeremy H.
中科院分区：
文献类型：
--
作者：
Le Brun, Anton P.;Holt, Stephen A.;Lakey, Jeremy H.
本文描述了一种膜蛋白阵列，该阵列在其恒定区结合免疫球蛋白 G，同时使可变区自由结合抗原。该阵列的支架是来自大肠杆菌的外膜蛋白 A (tOmpA) 的跨膜结构域，经过改造，通过单个半胱氨酸残基在金表面组装为定向单层。其他蛋白质结构域可以融合到支架的 N 和 C 末端。在本研究中，我们使用与金黄色葡萄球菌蛋白 A (ZZctOmpA) 的两个 Z 结构域融合的循环排列的 ctOmpA 来创建免疫球蛋白 G 结合阵列。通过圆二色光谱评估工程蛋白质的溶液结构。使用表面等离子体共振和中子反射测量阵列的组装、抗体的附着和抗原结合。与小鼠 IgG2 相比，来自兔子的多克隆 IgG 与 ZZctOmpA 的结合非常牢固，并且免疫球蛋白的解离速度足够慢，足以对带有抗原的组装层进行中子反射研究。使用磁性和同位素对比定义了完整的逐层模型，该模型揭示了 223 A 高层包含垂直方向的抗体。 (C) 2011 Elsevier Ltd. 保留所有权利。
This paper describes a membrane protein array that binds immunoglobulin G at its constant regions whilst leaving the variable regions free to bind antigen. The scaffold of the array is the transmembrane domain of outer membrane protein A (tOmpA) from Escherichia colt engineered to assemble as an oriented monolayer on gold surfaces via a single cysteine residue. Other protein domains can be fused to the N and C termini of the scaffold. In this study we use circularly permuted ctOmpA fused to two Z domains of Staphylococcus aureus protein A (ZZctOmpA) to create the immunoglobulin G-binding array. The solution structure of the engineered proteins was assessed by circular dichroism spectroscopy. Assembly of the array, attachment of antibodies and antigen binding were measured using surface plasmon resonance and neutron reflection. Compared to mouse IgG2, polyclonal IgG from rabbit bound very strongly to ZZctOmpA and the dissociation of the immunoglobulin was slow enough to allow neutron reflection studies of the assembled layer with antigen. Using both magnetic and isotopic contrasts a complete layer by layer model was defined which revealed that the 223 A high layer contains antibodies in an upright orientation. (C) 2011 Elsevier Ltd. All rights reserved.