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.
Keywords: 3′-Untranslated region (3′-UTR); Arabidopsis thaliana; Cell-free protein synthesis (CFPS); In vitro; Transcription factor (TF); Wheat germ extract (WGE).