Ribosome-Ribosome Dual Display

Protein-protein interactions underlie countless biological functions, motivating their analysis and engineering for fundamental inquiries and biotechnological applications. Unfortunately, many proteins of interest (POI) are difficult to express in conventional heterologous expression systems, impeding these efforts. Previous work has shown that a cell-free protein engineering method, ribosome display, can be used as an analytical tool to overcome this technological hurdle by tethering the recalcitrant POI to a ribosome, which acts as a large solubility tag that disfavors aggregation and enhances folding.¹ By panning the displayed POI and mutational variants against its recombinantly expressed target, a functional epitope of the POI was elucidated.

What if the POI and its protein target are both recalcitrant to heterologous expression? We present here the development of ribosome-ribosome dual display (R²D²) to address this issue. In R²D², the POI and its potential interacting partner(s) are separately displayed in ribosomal complexes, each of which also tethers the mRNA encoding its protein. The ribosomal complexes are co-incubated and the bound complexes are selectively recovered, allowing for identification of specific binding signals as a function of POI changes (e.g., subdomain or mutational analysis). Here, we show proof-of-concept results for R²D² using a model protein-protein interaction, and we are extending our approach to recalcitrant proteins. This approach should enable characterization and engineering of biotechnologically significant proteins that are difficult to express.

¹ Schimmele and Plückthun. J Mol Biol. 352:229-241 (2005).