High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.
Current cell-free protein expression systems are capable of synthesizing proteins with high speed and accuracy; however, the yields are low due to their instability over time. Escherichia coli based systems are not always sufficient for expression of eukaryotic proteins. This report reviews a high-throughput protein production method based on the cell-free system prepared from eukaryote, wheat embryos. We first demonstrate a method for preparation of this extract that exhibited a high degree of stability and activity. To maximize translation yield and throughput, we address and resolve the following issues: (1) optimization of the ORF flanking regions; (2) PCR-based generation of DNA for mRNA production; (3) expression vectors for large-scale protein production; and (4) a translation reaction that does not require a membrane. The combination of these elemental processes with robotic automation resulted in high-throughput protein synthesis.
Endo
Yaeta
Y
Cell-Free Science and Technology Research Center, and the Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan. yendo@en3.ehime-u.ac.jp
Sawasaki
Tatsuya
T
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D029681:Arabidopsis Proteins / Q000096:biosynthesis / Q000235:genetics; D002474:Cell-Free System; D004274:DNA, Recombinant / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D015894:Genome, Human; D018745:Genome, Plant; D023281:Genomics; D006801:Humans; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D040901:Proteomics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D012371:Robotics; D014908:Triticum / Q000235:genetics* / Q000378:metabolism*
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D029681:Arabidopsis Proteins; D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins