ADAPT-M: A workflow for rapid, quantitative in vitro measurements of enriched protein libraries
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Abstract
Protein-protein interactions underpin most cellular interactions, and engineered binders present powerful tools for probing biology and developing novel therapeutics. One bottleneck in binder generation is the scalable, quantitative characterization of these interactions. We present ADAPT-M ( A ffinity D etermination by A daptation of P ro T ein binders for M icrofluidics), a streamlined workflow that connects yeast surface display (YSD) with in vitro affinity and kinetic measurements using the high-throughput STAMMPPING microfluidic platform. ADAPT-M quantifies K d s and dissociation kinetic parameters for hundreds of enriched protein variants in under one week without requiring hands-on protein purification. We applied ADAPT-M to a computationally designed library targeting the SARS-CoV-2 Omicron BA.1 receptor binding domain, successfully recovering and measuring K d s for most highly enriched YSD variants. Measurements correlate strongly with biolayer interferometry and yeast titration assays. ADAPT-M further enabled selection of lead candidates for structural and mutational analysis, which revealed designed paratopes were preserved despite binding to off-target epitopes. By bridging YSD screening and in vitro validation, ADAPT-M accelerates protein binder discovery and supports data-driven protein engineering.
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fast dissociators
It would be interesting to see how these different categories of dissociators compare to your categories of Kd's, since they tell different sides of the story. It would be cool to see how you are adding another element of data here (and how you synthesized all of it to make a decision on what to characterize, or how one might move forward with that data).
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rebindin
I really like how you are able to isolate the binding event to a single spot. That makes detection so much easier. If you could track dissociation in real time (like you can in BLI), you wouldn't need to 'trap' and then image again. I wonder if some approach like that is possible. Either imaging many wells in real time, or imaging one at a time but controlling the flow of one chamber at a time so you can observe the whole span of the reaction.
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patterns)
Totally agree. It makes sense that the proteins that are expressed in a cell free system may behave differently than ones expressed in yeast.
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compressed
Do you have a sense of the protein aggregation level or stability of your gfp-tagged proteins? It seems like if they are expressed and subsequently bound to one part of the chip, many could be aggregated and it might not be clear (though perhaps you could visually see it).
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imaged
I wonder how you control for a set amount of dissociation - after you flow out any protein that has dissociated, you have disrupted equilibrium, and more will dissociate to make up for it, so you're kind of fighting this never-ending dissociation battle.
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