title: Coupled transcription and translation from polymerase chain reaction-amplified DNA in Drosophila Schneider 2 cell-free system.
abstract: Cell-free protein synthesis is one of the best methods to express recombinant proteins efficiently. We have developed a transcription and translation coupled (TnT) cell-free system from Drosophila Schneider 2 (S2) cells. To improve the translational efficiency of messenger RNA (mRNA) lacking a 5'-cap structure, we employed the sequence of the 5'-untranslated region derived from baculovirus p10 mRNA. By using this sequence, we established a polymerase chain reaction fragment-based TnT system. FLAG-tagged human Argonaute2 and TRBP2 were successfully synthesized within 90 min in a reaction volume of less than 10 microl and were detected by the FLAG antibody.
authors.affiliation: RIKEN Systems and Structural Biology Center, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Motoaki
authors.initials: M
authors.lastname: Wakiyama
authors.forename: Yoko
authors.initials: Y
authors.lastname: Kaitsu
authors.forename: Tomoko
authors.initials: T
authors.lastname: Matsumoto
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D020121:5' Untranslated Regions; D000818:Animals; D060565:Argonaute Proteins; D002474:Cell-Free System; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism; D004330:Drosophila / Q000166:cytology / Q000235:genetics* / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D020121:5' Untranslated Regions; D060565:Argonaute Proteins; D004247:DNA; D015852:Eukaryotic Initiation Factor-2; D009842:Oligopeptides; D010455:Peptides; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D014764:Viral Proteins; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human; C078072:p10 protein, Baculovirus; C085706:FLAG peptide