A cell-free protein synthesis system for high-throughput proteomics.
We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.
Sawasaki
Tatsuya
T
Department of Applied Chemistry, Faculty of Engineering, and Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan.
Ogasawara
Tomio
T
Morishita
Ryo
R
Endo
Yaeta
Y
D002474:Cell-Free System; D017931:DNA Primers; D030562:Databases, Protein; D004798:Enzymes / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D010940:Plant Proteins / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D011506:Proteins / Q000235:genetics*; D040901:Proteomics / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D014908:Triticum
D017931:DNA Primers; D004798:Enzymes; D010940:Plant Proteins; D011506:Proteins; D012333:RNA, Messenger