Egger M, Heyn S P, Gaub H E
Technische Universität München, Physikdepartment E22, Garching, FRG.
Biophys J. 1990 Mar;57(3):669-73. doi: 10.1016/S0006-3495(90)82586-8.
A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.
在气-水界面形成了定向抗体片段的二维图案，并将其转移到固体支持物上。针对半抗原二硝基苯基（DNP）的单克隆抗体的Fab'片段通过亲水性间隔物与磷脂囊泡共价连接。在气-水界面允许与这些囊泡达到平衡的单分子脂质-蛋白质层形成。将单分子层与囊泡相分离，并转移到Langmuir-Blodgett槽中。通过冷却和压缩，将先前均匀的脂质-蛋白质膜驱动到二维相分离中，从而产生富含蛋白质的区域和主要由脂质组成的第二相。以保留蛋白质-脂质图案的方式将该膜转移到固体支持物上。然后使用显微荧光测定法定量两种不同分离相的特异性以及结合活性的对比度。与荧光素标记的牛血清白蛋白（BSA）缀合的DNP实际上与脂质区域没有结合，但在富含蛋白质的区域中结合的DNP-BSA与Fab'-脂质的比例大于50%，证明Fab'部分保留了其生物活性。这表明本文提出的技术非常适合用给定生物功能的图案修饰不同的固体表面。对单分子层中组分的横向堆积和取向进行的选择性控制使其成为重建支持的脂质-蛋白质膜的通用工具，也可能为膜上蛋白质的二维结晶开辟新途径。
