Proteomic Approach to the Separation and Identification of Rice (Oryza sativa L.) Proteins from Suspension Cell Culture

초록

Proteome analysis is a powerful tool for separating whole protein mixture form cell extract and identifying new proteins in relation to cell metabolism. In the last few years, proteomics has been dynamically studied and lots of functional proteins have been already identified in some fields such as human and E. coli using sufficient genome database. Two-dimensional electrophoresis in proteomics is the most useful technique for the high-resolution protein separation. Although there are significant results from many organisms, very few attempts have been made for the proteomics of plant cells. Rice cells are a useful material for the study of plant proteomics because their genetic information is being actively investigated. Rice cells are, above all, utilized as a host for producing recombinant proteins, therefore protein separation from both rice suspension cells and culture media is essential for studying the relationship between host proteins and a target recombinant protein. In this study, we optimized the methods of sample preparation form rice suspension cells and cultured media, for 2-DE system. Proteins from cell extracts containing high-level interfering substances were prepared with phenol extraction and ammonium acetate precipitation method which was more effective than TCA and acetone precipitation method. Desalting of media sample preparation is an important step for successful IEF therefore salts were sorted out by 5？e buffer exchange with 10 kDa filter membrane. About 700 and 200 spots were detected in rice suspension cells and media, respectively. Separation in the first dimension was performed using two pH ranges : pH 4-7, pH 6-11 and SDS-PAGE was accomplished with 9~18% gradient gel.