受賞講演
- 第3日 5月17日（木） 17:45～17:55 A会場（オービットホール）
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3A-AW-1745 PDF
コムギ無細胞合成法を利用した標準ペプチド多重共発現系の開発と定量プロテオミクスへの応用
Wheat germ cell-free synthesis (WGCFS) is a powerful method that provides the highest translation efficiency among eukaryotic cell-free systems. In vitro translation with WGCFS is a superior method for efficient incorporation of stable isotope-labeled amino acids into proteins. We developed a high-throughput technique for synthesis of stable isotope-labeled standard proteins using WGCFS. That methodology gave highly efficient isotope labeling of > 99%, and was effective for synthesis of standard proteins, including transmembrane proteins, which are difficult targets when using the cell-based approach. To extend the opportunities for large-scale absolute proteome quantification, we then focused on the cell-free synthesis of QconCAT, an artificial protein consisting concatenated peptides from different sources. Its stable isotope-labeled form provides multiplexing standards for quantitation of endogenous targeted proteins. WGCFS has a high success rate for expression of QconCATs, especially for some that are difficult to express in E. coli. The multiple protein biosynthesis capability of WGCFS allows simultaneous synthesis of 150 QconCATs in 2 days. This innovative technology for synthesis of peptide standards is designated “MEERCAT” (Multiplexed Efficient Expression of Recombinant QconCATs). MEERCAT will enable rapid construction of large-scale standard peptide libraries in individual laboratories and accelerate the quantification of proteomes in various model organisms.