PUREfrex^® is a cell-free protein synthesis system based on the PURE system, which is reconstituted only from factors involved in protein synthesis in E. coli.
Due to the improvement of the reaction composition, the synthesis efficiency has increased up to 1 mg/mL, but it varies significantly depending on the target protein.
In this presentation, we will report our findings on the influence of the 5'UTR and N-terminal region of ORF on the protein synthesis efficiency.

(1)    5'UTR
To confirm the necessity of each region in the 5'UTR derived from T7 phage currently in use, we compared the fluorescence of sfGFP synthesized from its template DNA with different 5′UTR. As a result, when either the AT-rich region or the Shine-Dalgarno (SD) sequence was completely removed, the fluorescence decreased to less than 10%. This result indicates that not only the SD sequence but also the AT-rich region in the 5'UTR is important for the efficient translation in PUREfrex^®.

(2)    N-terminal region of ORF
It was reported that the N-terminal region of ORF also influences the protein synthesis efficiency. First, for ten amino acids immediately below the start codon, the synthesis efficiency was higher when AT-rich codons were used than when codons commonly used in E. coli were used. For some proteins, changing a few codons in the N-terminal region increased synthesis efficiency more than 10-fold. Second, amino acids in the N-terminal region also affected the synthesis efficiency, especially at low temperatures. For example, replacing glycine at position 4 of sfGFP with tyrosine increased synthesis efficiency 8-fold at 23°C. These results indicate that small differences in the N-terminal sequence can cause large fluctuations in the synthesis efficiency.