de Lorimier Robert M, Tian Yaji, Hellinga Homme W
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Protein Sci. 2006 Aug;15(8):1936-44. doi: 10.1110/ps.062261606. Epub 2006 Jul 5.
Development of biosensor devices typically requires incorporation of the molecular recognition element into a solid surface for interfacing with a signal detector. One approach is to immobilize the signal transducing protein directly on a solid surface. Here we compare the effects of two direct immobilization methods on ligand binding, kinetics, and signal transduction of reagentless fluorescent biosensors based on engineered periplasmic binding proteins. We used thermostable ribose and glucose binding proteins cloned from Thermoanaerobacter tengcongensis and Thermotoga maritima, respectively. To test the behavior of these proteins in semispecifically oriented layers, we covalently modified lysine residues with biotin or sulfhydryl functions, and attached the conjugates to plastic surfaces derivatized with streptavidin or maleimide, respectively. The immobilized proteins retained ligand binding and signal transduction but with adversely affected affinities and signal amplitudes for the thiolated, but not the biotinylated, proteins. We also immobilized these proteins in a more specifically oriented layer to maleimide-derivatized plates using a His(2)Cys(2) zinc finger domain fused at either their N or C termini. Proteins immobilized this way either retained, or displayed enhanced, ligand affinity and signal amplitude. In all cases tested ligand binding by immobilized proteins is reversible, as demonstrated by several iterations of ligand loading and elution. The kinetics of ligand exchange with the immobilized proteins are on the order of seconds.
生物传感器设备的开发通常需要将分子识别元件整合到固体表面，以便与信号检测器连接。一种方法是将信号转导蛋白直接固定在固体表面。在这里，我们比较了两种直接固定方法对基于工程化周质结合蛋白的无试剂荧光生物传感器的配体结合、动力学和信号转导的影响。我们分别使用了从腾冲嗜热厌氧菌和海栖热袍菌克隆的热稳定核糖和葡萄糖结合蛋白。为了测试这些蛋白质在半特异性定向层中的行为，我们用生物素或巯基功能共价修饰赖氨酸残基，并将缀合物分别连接到用链霉亲和素或马来酰亚胺衍生化的塑料表面。固定化的蛋白质保留了配体结合和信号转导能力，但硫醇化蛋白质的亲和力和信号幅度受到不利影响，而生物素化蛋白质则不受影响。我们还使用在其N端或C端融合的His(2)Cys(2)锌指结构域，将这些蛋白质以更特异性的定向层固定在马来酰亚胺衍生化的平板上。以这种方式固定的蛋白质要么保留，要么显示出增强的配体亲和力和信号幅度。在所有测试的情况下，固定化蛋白质的配体结合都是可逆的，这通过配体加载和洗脱的几次迭代得到证明。配体与固定化蛋白质交换的动力学在秒的量级。
