Abstract
Biotic and abiotic stimuli induce profound transcriptional reprograming in plants through sophisticated regulation of transcription factors (TFs). Recombinant proteins of TFs play an important role in unveiling their molecular functions. Cell-free protein synthesis (CFPS) system from wheat germ has been developed as one of the most efficient protein synthesis platforms. However, preparation of linear DNA templates for in vitro transcription is time-consuming and laborious. Here, we describe a versatile method for in vitro transcription and translation of the wheat germ CFPS system. Our two-step PCR method enables researchers to generate a variety of transcription templates from a single plasmid including fusion proteins of an N- or C-terminal tag and truncated proteins. Thus, this method supports a rapid and high-throughput expression of proteins for a large-scale proteomics analysis.
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References
Riechmann JL, Heard J, Martin G et al (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110
Skelly MJ, Frungillo L, Spoel SH (2016) Transcriptional regulation by complex interplay between post-translational modifications. Curr Opin Plant Biol 33:126–132
Hill K (2015) Post-translational modifications of hormone-responsive transcription factors: the next level of regulation. J Exp Bot 66:4933–4945
Spoel SH, Tada Y, Loake GJ (2010) Post-translational protein modification as a tool for transcription reprogramming. New Phytol 186:333–339
Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:172
Baneyx F, Mujacic M (2004) Recombinant protein folding and misfolding in Escherichia coli. Nat Biotechnol 22:1399–1408
Katzen F, Chang G, Kudlicki W (2005) The past, present and future of cell-free protein synthesis. Trends Biotechnol 23:150–156
Chong S (2014) Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications. Curr Protoc Mol Biol 108:16.30.1–11
Carlson ED, Gan R, Hodgman CE et al (2012) Cell-free protein synthesis: applications come of age. Biotechnol Adv 30:1185–1194
Zemella A, Thoring L, Hoffmeister C et al (2015) Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. Chembiochem 16:2420–2431
O’Malley RC, Huang SS, Song L et al (2016) Cistrome and Epicistrome features shape the regulatory DNA landscape. Cell 165:1280–1292
Takahashi H, Ozawa A, Nemoto K et al (2012) Genome-wide biochemical analysis of Arabidopsis protein phosphatase using a wheat cell-free system. FEBS Lett 586:3134–3141
Sawasaki T, Ogasawara T, Morishita R et al (2002) A cell-free protein synthesis system for high-throughput proteomics. Proc Natl Acad Sci U S A 99:14652–14657
Takai K, Sawasaki T, Endo Y (2010) Practical cell-free protein synthesis system using purified wheat embryos. Nat Protoc 5:227–238
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Nomoto, M., Tada, Y. (2018). Cell-Free Protein Synthesis of Plant Transcription Factors. In: Yamaguchi, N. (eds) Plant Transcription Factors. Methods in Molecular Biology, vol 1830. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8657-6_20
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DOI: https://doi.org/10.1007/978-1-4939-8657-6_20
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-8657-6
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