Cell-Free Transcription and Translation Tether Expressed Peptides to Their Encoding Circular DNA

We report a novel cell-free technology, ICED (Intra-Circular Expression and Display), for displaying an expressed protein on its encoding circular DNA. The recovered circular DNA, enriched by affinity-based screening of nascent protein, can be directly amplified using RCR, the reconstituted <i>E. coli</i> replication-cycle reaction. Unlike CIS display, which requires the replication initiator RepA and cis-acting elements including the <i>oriR</i>, the cognate binding site of RepA from the R1 plasmid, ICED does not depend on such a specific protein or DNA elements. The display is abolished by linearization of template DNA, inserting a transcription terminator, or treatment with RNaseA as well as puromycin. Its efficiency is enhanced by the addition of magnesium in the selection step. These suggest that the expressed protein remains anchored to the circular DNA via a transcription-translation (TX-TL) complex involving RNA polymerase, mRNA, and ribosome. This previously unrecognized linkage offers new insight into the mechanistic interface in TX-TL. Notably, the system is compatible not only with crude extracts but also with the reconstituted PURE system composed of <i>E. coli</i> or T7 RNA polymerase and purified translation factors. By directly reusing the selected RCR product for subsequent rounds, we achieved 10⁸-fold enrichment by two rounds, surpassing the performance of conventional display platforms. ICED thus provides a more efficient and straightforward platform for cell-free display.