PUREfrex® is a reconstituted cell-free protein synthesis system based on the PURE system, composed solely of factors essential for protein synthesis in Escherichia coli (E. coli). Due to its highly adjustable reaction composition, PUREfrex® is well-suited for the amber suppression method, in which non-canonical amino acid (ncAA) is incorporated at an amber stop codon (UAG) using a suppressor tRNA charged with the ncAA. However, detailed optimization of the reaction conditions for amber suppression using the PURE system has not been thoroughly investigated.

In this study, we explored the optimal conditions for amber suppression using PUREfrex®. As a model protein, we used a construct in which S-tag (KETAAAKFERSQHMDS) was fused to the N-terminus of E. coli dihydrofolate reductase (DHFR). We synthesized the fusion protein in the presence of an amber suppressor tRNA with BODIPY FL-aminophenylalanine (BFLAF) from the template DNA containing an amber codon within S-tag. The suppression efficiency was evaluated by measuring the fluorescence of BODIPY in the synthesized protein.

First, we examined the optimal position of an amber codon within S-tag. As a result, the suppression efficiency was high when an amber codon was introduced at positions T7 and F12. Next, using these constructs, we optimized the concentration of some components and BFLAF-charged suppressor tRNA in the reaction mixture. Unexpectedly, we found that the addition of release factor 1 (RF1), which recognizes an amber codon, improved the suppression efficiency. In addition, we found that the codon immediately preceding an amber codon significantly affected the suppression efficiency and the yield of full-length protein.

We believe these results provide valuable insights for amber suppression using PUREfrex®.