High-throughput, genome-scale protein production method based on the wheat germ cell-free expression system.
Cell-free protein synthesis systems can synthesize proteins with high speed and accuracy, but produce only a low yield because of their instability over time. Here we review our recent advances in a cell-free protein synthesis system prepared from wheat embryos. We first addressed and resolved the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins. We found that conventional wheat germ extracts contained the RNA N-glycosidase tritin and other inhibitors such as thionin, ribonucleases, deoxyribonucleases, and proteases that originate from the endosperm and inhibit translation. Extensive washing of wheat embryos to eliminate endosperm contaminants has resulted in extracts with a high degree of stability and activity. To maximize the translation yield and throughput of the system, we then focused on developing the following issues: optimization of the ORF flanking regions, a new strategy to construct PCR-generated DNAs for screening, and design of an expression vector for large-scale protein production. The resulting system achieves high-throughput expression, with a PCR-directed system at least 50 genes that can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Under the dialysis mode of reaction, the system with the expression vector can maintain productive translation for 14 days. The cell-free system described here bypasses most of the biological processes and lends itself to robotic automation for high-throughput expression of genetic information, thus opening up many possibilities in the post-genome era.
Endo
Yaeta
Y
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama790-8577, Japan. yendo@en3.ehime-u.ac.jp
Sawasaki
Tatsuya
T
D002474:Cell-Free System / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000502:physiology; D018506:Gene Expression Regulation, Plant / Q000502:physiology*; D018745:Genome, Plant; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering / Q000379:methods*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis*; D014908:Triticum / Q000196:embryology / Q000235:genetics* / Q000378:metabolism*
D010940:Plant Proteins; D011994:Recombinant Proteins