High-throughput Screening of Proteins by Electrochemical Protein Chip

随着人类基因组测序计划接近完成，人们的注意力现在转移到了蛋白质或蛋白质组的分析上。在这方面，蛋白质芯片被认为是研究蛋白质表达的高通量手段。蛋白质芯片是由固定在底物表面的各种蛋白质制成的。本项目的目的是在我们对电化学DNA芯片的研究中获得的知识基础上开发一种蛋白质芯片。为了实现这一目的，我们通过金-硫键将携带半胱氨酸残基的蛋白质固定在金电极上，构建了一种蛋白质芯片，并尝试用它建立了一种电化学分析系统。用电化学方法研究了不同电荷的多肽在金电极上的固定及其与二茂铁甲酸的相互作用。二茂铁羧酸与阳离子多肽im…强结合在接下来的实验中，利用Fmoc化学合成的多肽合成器合成了能区分单链和双链DNA的Rec A蛋白及其功能结构域L2和Helix G，并测试了它们与单链DNA的相互作用是否可以被电化学监测。这些多肽被固定在单独的电极上，并允许与携带有电化学活性的二茂铁部分的单链或双链DNA相互作用。该系统一旦建立，将有助于筛选DNA结合肽或蛋白质。电化学和石英晶体微天平实验表明，蛋白质的L2部分对于与DNA碱基的相互作用是重要的，而螺旋G部分有助于区分单链和双链DNA。这些结果为电化学蛋白质芯片的发展开辟了新的前景。此外，使用扫描电化学显微镜应该可以高通量地筛选各种其他蛋白质。较少

By the near completion of the human genome sequencing project, attention is now shifting toward the analysis of proteins or proteoms. In this respect, protein chips are regarded as a high-throughput means of studying protein expression. Protein chips are made of various proteins immobilized on the surface of a substrate. The aim of this project is to develop a protein chip based on the knowledge obtained from our study of electrochemical DNA chips. To realize this purpose, we constructed a protein chip by immobilizing a protein carrying a cysteine residue on the gold electrode through the gold-sulfur linkage and tried to establish an electrochemical analysis system with it.First of all, we verified in a preliminary experiment with peptides that this system works as designed. Peptides carrying different charges immobilized on a gold electrode and their interaction with ferrocenecarboxylic acid was studied electrochemically. Ferrocenecarboxylic acid bound strongly to cationic peptides im … More mobilized on the electrode as proven by an increase in the redox current due to the ferrocene, whereas it failed to bind to anionic peptides because of electrostatic repulsion.In the next experiment, Rec A protein, known to discriminate single and double stranded DNA, and its functional domains, L2 and Helix G peptides, were synthesized by the peptide synthesizer with Fmoc chemistry and tested whether their interaction with single stranded DNA can be monitored electrochemically. These peptides were immobilized on the individual electrodes and were allowed to interact with single or double stranded DNA carrying an electrochemically active ferroncene moiety. This system, once established, should be useful for the screening of DNA binding peptides or proteins. Electrochemical as well as Quartz Crystal Microbalance experiments suggested that the L2 part of the protein is important for the interaction with nucleic bases of DNA and the Helix G part contributes to the discrimination between single and double stranded DNAs. These results opened up a new vista for the development of an electrochemical protein chip. Furthermore, high-throughput screening of various other proteins should be possible by using the scanning electrochemical microscope. Less