PT  - JOURNAL ARTICLE
AU  - Curtis J Layton
AU  - Peter L McMahon
AU  - William J Greenleaf
TI  - Large-scale, quantitative protein assays on a high-throughput DNA sequencing chip
AID  - 10.1101/342808
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 342808
4099  - http://biorxiv.org/content/early/2018/06/14/342808.short
4100  - http://biorxiv.org/content/early/2018/06/14/342808.full
AB  - 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 a widely-used high-throughput sequencing chip to display an immense diversity of ribosomally-translated proteins and peptides, then carry out fluorescence-based functional assays directly on this flow cell. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3×104 variant FLAG peptides, discovering non-additive effects of combinations of mutations, as well as a “superFLAG” epitope variant. We also measured the enzymatic activity of 1.56×105 molecular variants of full-length of human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates linked to amino acid sequence perturbations revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively-cooperative interactions with histidine residues.One Sentence Summary We have adapted a widely-used Illumina high-throughput sequencing chip to display an immense diversity of ribosomally-translated proteins and peptides on which we directly carry out fluorescence-based functional assays, demonstrating that a single, widely-available high-throughput platform can perform both sequencing-by-synthesis and protein assays, enabling quantitative exploration of the relationship between protein and peptide sequence, structure, and function.