Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip

High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a “superFLAG” epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues. [Display omitted] •Generation of a massive peptide and protein array on a sequencing flow cell•Direct measurement of binding and catalysis for ∼105 protein mutational variants•Patterns of amino acid mutation and cooperativity were analyzed in high throughput•Discovery of high-avidity “superFLAG” epitope for the M2 anti-FLAG antibody By generating a massive peptide and protein array in situ on an Illumina sequencing flow cell with in vitro translation, Layton et al. demonstrate direct protein assays for enzyme function and antibody-peptide interactions across large libraries (∼105) of mutational variants on an automated platform.