Coupled transcription and translation from polymerase chain reaction-amplified DNA in Drosophila Schneider 2 cell-free system

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 mu l and were detected by the FLAG antibody. (C) 2010 Elsevier Inc. All rights reserved.