High-throughput Screening of Proteins by Electrochemical Protein Chip

电化学蛋白质芯片高通量筛选蛋白质

基本信息

批准号：
12555236
负责人：
TAKAGI Makoto
金额：
$ 8.77万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12555236/
关键词：
protein chip electrochemistry gold electrode electrochemical chip ferrocene L2-G peptide L2 peptide Helix G peptide 静電的相互作用

项目摘要

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

通过人类基因组测序项目的近乎完整的注意力，Tein芯片被视为研究蛋白质表达的高通量。通过固定在金电极上携带半胱氨酸残基的蛋白质，并试图与IT建立电化学分析系统。首先，与系统相互作用的所有初步经验。在电极上由氧化还原电流证明，由于甲状腺素的厌恶率证明。在下一个实验中，rec rec rec蛋白质（已知会区分单个和双重链DNA及其功能结构域，及其功能结构域由肽合成器与FMOC化学和FMOC化学和测试了与单个链DNA的相互作用是否可以固定在单个电极上，并被允许到ITH单链或双链DNA携带的DNA活性铁烯酮部分，而石英晶体微体验实验表明L2是ITH的Eximent Ith ITH ITH ITH ITH ITH ITH ITH ITHINT Heli X G部分的核碱基有助于单链DNA之间的公开，以开发新的远景，以开发电化学蛋白质