PUREfrex® is a build up cell-free protein synthesis system that consists of the factors involved in transcription and translation in E. coli, and with the improvement of the reaction mixture, the synthesis amount of the product has been increased up to about 1 mg/ml.

We are exploring not only reaction mixture but also appropriate DNA sequence or structure for better translation efficiency in PUREfrex®. For example, we reported that AT rich codons instead of most frequently used codons in E.coli at N-terminal region of ORF led to higher productivity, and 5’UTR should have AT rich region as well as ribosome binding sequence (SD) because removing AT rich region decreased the translation efficacy about 1/10.

Based on the findings so far, bicistronic template DNA having two genes were investigated in this poster. The template DNA was prepared coding ORF of fluorescence protein mCherry followed by AT-rich region, SD and ORF of GFP, and each fluorescence was detected. The result showed higher fluorescence of mCherry and lower fluorescence of GFP comparing to that with separate template DNAs for each. Removing SD at upstream of GFP led to almost no fluorescence of GFP, but removing AT rich region didn’t have impact on synthesis of GFP. As a result, it was confirmed that bicistronic template DNA needed only SD at the upstream of second ORF.