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

Analytical Biochemistry
Motoaki WakiyamaShigeyuki Yokoyama

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.

References

Jan 19, 2000·Proceedings of the National Academy of Sciences of the United States of America·K MadinY Endo
Jun 29, 2000·Annual Review of Biochemistry·A C GingrasN Sonenberg
Jul 21, 2004·Journal of Structural and Functional Genomics·Takanori KigawaShigeyuki Yokoyama
Mar 1, 2005·Trends in Biotechnology·Federico KatzenWieslaw Kudlicki
Nov 18, 2005·Protein Expression and Purification·Satoshi MikamiHiroaki Imataka
Apr 4, 2006·Biochemical and Biophysical Research Communications·Motoaki WakiyamaShigeyuki Yokoyama
Jan 2, 2008·Journal of Structural and Functional Genomics·Takashi YabukiShigeyuki Yokoyama

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Citations

May 1, 2013·Current Opinion in Structural Biology·Frank Bernhard, Yuzuru Tozawa

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