title: A highly efficient and robust cell-free protein synthesis system prepared from wheat embryos: plants apparently contain a suicide system directed at ribosomes.
abstract: Current 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 describe the preparation of a highly efficient but also robust cell-free system from wheat embryos. We first investigated the source of the instability of existing systems in light of endogenous ribosome-inactivating proteins and found that ribosome inactivation by tritin occurs already during extract preparation and continues during incubation for protein synthesis. Therefore, we prepared our system from extensively washed embryos that are devoid of contamination by endosperm, the source of tritin and possibly other inhibitors. In a batch system, we observed continuous translation for 4 h, and sucrose density gradient analysis showed formation of large polysomes, indicating high protein synthesis activity. When the reaction was performed in a dialysis bag, enabling the continuous supply of substrates together with the continuous removal of small byproducts, translation proceeded for >60 h, yielding 1-4 mg of enzymatically active proteins, and 0.6 mg of a 126-kDa tobacco mosaic virus protein, per milliliter of reaction volume. Our results demonstrate that plants contain endogenous inhibitors of translation and that after their elimination the translational apparatus is very stable. This contrasts with the common belief that cell-free translation systems are inherently unstable, even fragile. Our method is useful for the preparation of large amounts of active protein as well as for the study of protein synthesis itself.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Madin
authors.forename: T
authors.initials: T
authors.lastname: Sawasaki
authors.forename: T
authors.initials: T
authors.lastname: Ogasawara
authors.forename: Y
authors.initials: Y
authors.lastname: Endo
mesh_terms: D002474:Cell-Free System; D049452:Green Fluorescent Proteins; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification; D010940:Plant Proteins / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism; D012639:Seeds / Q000737:chemistry / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics; D014027:Tobacco Mosaic Virus / Q000737:chemistry; D014908:Triticum / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D008164:Luminescent Proteins; D010936:Plant Extracts; D010940:Plant Proteins; D011506:Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014764:Viral Proteins; C031676:tritin protein, Triticum aestivum; D049452:Green Fluorescent Proteins; D008156:Luciferases; D013762:Tetrahydrofolate Dehydrogenase