Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, Elche, Spain ; Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
Instituto Universitario de Electroquímica. Universidad de Alicante, Alicante, Spain.
PLoS One. 2014 Jan 31;9(1):e87995. doi: 10.1371/journal.pone.0087995. eCollection 2014.
We have developed a general method for the specific and reversible immobilization of proteins fused to the choline-binding module C-LytA on functionalized graphite electrodes. Graphite electrode surfaces were modified by diazonium chemistry to introduce carboxylic groups that were subsequently used to anchor mixed self-assembled monolayers consisting of N,N-diethylethylenediamine groups, acting as choline analogs, and ethanolamine groups as spacers. The ability of the prepared electrodes to specifically bind C-LytA-tagged recombinant proteins was tested with a C-LytA-β-galactosidase fusion protein. The binding, activity and stability of the immobilized protein was evaluated by electrochemically monitoring the formation of an electroactive product in the enzymatic hydrolysis of the synthetic substrate 4-aminophenyl β-D-galactopyranoside. The hybrid protein was immobilized in an specific and reversible way, while retaining the catalytic activity. Moreover, these functionalized electrodes were shown to be highly stable and reusable. The method developed here can be envisaged as a general, immobilization procedure on the protein biosensor field.
我们开发了一种通用方法，用于将融合了胆碱结合模块 C-LytA 的蛋白质特异性且可逆地固定在功能化石墨电极上。通过重氮化学将石墨电极表面进行修饰，引入羧基，随后用于锚定由 N,N-二乙基亚乙基二胺基团（作为胆碱类似物）和乙醇胺基团（作为间隔物）组成的混合自组装单层。通过用 C-LytA-β-半乳糖苷酶融合蛋白测试制备的电极特异性结合 C-LytA 标记的重组蛋白的能力。通过电化学监测合成底物 4-氨基苯基 β-D-半乳糖吡喃糖苷的酶水解过程中形成的电活性产物来评估固定化蛋白的结合、活性和稳定性。该杂交蛋白以特异性和可逆的方式固定化，同时保留了催化活性。此外，这些功能化电极显示出高度的稳定性和可重复使用性。这里开发的方法可以被设想为蛋白质生物传感器领域的一种通用的固定化程序。
