Amplicon/Protein Bead Display enables quantitative in vitro biochemistry at scale

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Scalable methods for protein library production and characterization are crucial for developing predictive models of protein sequence-function relationships.
• Current techniques often face limitations in throughput and efficiency for large-scale quantitative analysis.

Purpose of the Study:

• To present Amplicon/Protein Bead Display (APB-Display), a novel platform for high-throughput *in vitro* protein variant expression and characterization.
• To demonstrate the capability of APB-Display for quantifying protein expression levels and binding affinities at scale.

Main Methods:

• APB-Display utilizes particle-templated emulsification to create hydrogel beads displaying both protein variants and their encoding DNA.
• Quantitative binding affinities (Kd) were determined using titration curves (APB-TiteSeq) and single-concentration measurements (APB-SortSeq) combined with neural network denoising.
• High-throughput screening was performed using FACS sorting and sequencing.

Main Results:

• APB-Display can produce and purify over 100,000 protein variants *in vitro* within a single day.
• Simultaneous quantification of expression and binding affinities for over 18,000 FLAG epitope variants was achieved in 3 days using APB-TiteSeq.
• APB-SortSeq yielded quantitative affinities for over 88,000 variants, revealing insights into sequence-dependent interactions.

Conclusions:

• APB-Display offers an accessible and scalable platform for quantitative *in vitro* biochemistry, requiring only standard laboratory equipment.
• The method significantly accelerates the generation of standardized datasets for training sequence-function models.
• APB-Display facilitates the discovery of complex molecular interactions and epistasis at an unprecedented scale.