Large-Scale, Quantitative Protein Assays on a High-Throughput DNA Sequencing Chip.
Mol Cell
; 73(5): 1075-1082.e4, 2019 03 07.
Article
em En
| MEDLINE
| ID: mdl-30849388
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.
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MEDLINE
Assunto principal:
Oligopeptídeos
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Análise Mutacional de DNA
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O(6)-Metilguanina-DNA Metiltransferase
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Análise de Sequência com Séries de Oligonucleotídeos
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Análise Serial de Proteínas
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Sequenciamento de Nucleotídeos em Larga Escala
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Anticorpos
Idioma:
En
Ano de publicação:
2019
Tipo de documento:
Article