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

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• High-throughput DNA sequencing is advanced, but protein function assays lack throughput and automation.
• Existing methods limit the scale and scope of protein functional analysis.

Purpose of the Study:

• To adapt a high-throughput sequencing platform for protein functional assays.
• To demonstrate simultaneous sequencing and protein analysis on a single platform.
• To explore protein variant interactions and enzymatic activities at scale.

Main Methods:

• Adapted an Illumina high-throughput sequencing chip for displaying diverse ribosomally translated proteins and peptides.
• Performed fluorescence-based functional assays directly on the sequencing flow cell.
• Quantified antibody-peptide binding and measured enzymatic activity of protein variants.

Main Results:

• Demonstrated a single platform for both DNA sequencing and protein assays.
• Identified a "superFLAG" epitope variant with enhanced antibody binding.
• Characterized 1.56 × 10^5 molecular variants of human O6-alkylguanine-DNA alkyltransferase (SNAP-tag).
• Revealed amino acid interaction networks, cooperativity, and structural proximity effects.

Conclusions:

• High-throughput sequencing platforms can be repurposed for large-scale protein functional assays.
• This approach enables discovery of novel protein variants and insights into protein interactions.
• The method significantly advances the capacity for protein engineering and functional characterization.