Abstract:
Typical methods for stable immobilization of proteins often involve time-consuming surface modification of silicon-based materials to enable specific binding, while the nonspecific adsorption method is faster but usually unstable. Herein, we fused a silica-binding protein, Si-tag, to target proteins so that the target proteins could attach directly to silica substrates in a single step, markedly streamlining the immobilization process. The adhesion force between the Si-tag and glass substrates was determined to be approximately 400-600 pN at the single-molecule level by atomic force microscopy, which is greater than the unfolding force of most proteins. The adhesion force of the Si-tag exhibits a slight increase when pulled from the C-terminus compared to that from the N-terminus. Furthermore, the Si-tag\'s adhesion force on a glass surface is marginally higher than that on a silicon nitride probe. The binding properties of the Si-tag are not obviously affected by environmental factors, including pH, salt concentration, and temperature. In addition, the macroscopic adhesion force between the Si-tag-coated hydrogel and glass substrates was ∼40 times higher than that of unmodified hydrogels. Therefore, the Si-tag, with its strong silica substrate binding ability, provides a useful tool as an excellent fusion tag for the rapid and mechanically robust immobilization of proteins on silica and for the surface coating of silica-binding materials.
摘要:
稳定固定蛋白质的典型方法通常涉及对硅基材料进行耗时的表面修饰,以实现特异性结合,而非特异性吸附方法速度更快,但通常不稳定。在这里,我们融合了二氧化硅结合蛋白,Si-tag,目标蛋白,以便目标蛋白可以在单个步骤中直接附着到二氧化硅底物上,显着简化固定过程。通过原子力显微镜确定,在单分子水平上,Si标签和玻璃基板之间的粘附力约为400-600pN。大于大多数蛋白质的展开力。当从C-末端拉时,与从N-末端拉时相比,Si-标签的粘附力显示出轻微的增加。此外,硅标签在玻璃表面上的粘附力略高于氮化硅探针上的粘附力。Si-tag的结合特性受环境因素的影响不明显,包括pH值,盐浓度,和温度。此外,Si-tag涂层水凝胶与玻璃基材之间的宏观粘合力比未改性水凝胶高40倍。因此,Si标签,以其强大的二氧化硅基质结合能力,提供了一种有用的工具,可作为出色的融合标签,用于将蛋白质快速且机械坚固地固定在二氧化硅上以及用于二氧化硅结合材料的表面涂层。