Although cell-free directed evolution methods have been used to engineer proteins for nearly two decades, selections on more complex phenotypes have largely remained in the domain of cell-based engineering approaches. Here, we review recent conceptual advances that now enable in vitro display of multimeric proteins, integral membrane proteins, and proteins with an expanded amino acid repertoire. Additionally, we discuss methodological improvements that have enhanced the accessibility, efficiency, and robustness of cell-free approaches. Coupling these advances with the in vitro advantages of creating exceptionally large libraries and precisely controlling all experimental conditions, cell-free directed evolution is poised to contribute significantly to our understanding and engineering of more complex protein phenotypes.

No abstract

Plant immune receptors are often difficult to express heterologously, hindering study of direct interactions between these receptors and their targets with traditional biochemical approaches. The cell-free method ribosome display (RD) enables expression of such recalcitrant proteins by keeping each nascent polypeptide chain tethered to its ribosome, which can enhance protein folding by virtue of its size and solubility. Moreover, in contrast to an in planta readout of receptor activity such as a hypersensitive response that conflates binding and signaling, RD enables direct probing of the interaction between plant immune receptors and their targets. Here, we demonstrate the utility of this approach using tomato recognition of Trichoderma viride ethylene-inducing xylanase (EIX) as a case study. Leveraging the modular nature of the tomato LeEIX2 and LeEIX1 leucine-rich repeat (LRR) receptors, we applied an entropy-informed algorithm to maximize the information content in our receptor segmentation RD experiments to identify segments implicated in EIX binding. Unexpectedly, two distinct EIX-binding hotspots were discovered on LeEIX2 and both hotspots are shared with decoy LeEIX1, suggesting that their contrasting receptor functions are not due to differential modes of ligand binding. Given that most plant immune receptors are thought to engage targets via their LRR sequences, this approach should be of broad utility in rapidly identifying their binding hotspots.