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 E. coli replication-cycle reaction.
Unlike CIS display, which requires the replication initiator RepA
and cis-acting elements including the oriR, 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 E. coli 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.