Dissecting enzyme function at scale using synergistic advances in microfluidics and genetic code expansion

Summary

This project aims to develop and apply a high-throughput microfluidic platform for the parallel expression, purification, and quantitative assay of over 1,000 noncanonical amino acid (ncAA)-harboring protein variants, specifically to understand the catalytic mechanisms of proton transfer in alanine racemase.

What they want

Leverage the high-throughput microfluidic enzyme kinetics (HT-MEK) platform to enable parallel expression, purification, and quantitative assay of more than 1,000 ncAA-harboring protein variants on a single microfluidic device. Collect over 10,000 gold-standard biochemical measurements of ncAA-containing proteins. Apply this technique to understand the catalytic mechanisms governing proton transfer at carbon in alanine racemase (AlaR), a pyridoxal 5’-phosphate (PLP)-dependent enzyme. Systematically and precisely perturb the electrostatic properties of critical catalytic residues in the active site of AlaR across 96 different enzyme variants using ncAAs. Investigate how active site interactions optimize proton transfer for high efficiency at neutral pH and achieve 10^6:1 regioselectivity among competing pathways for reprotonation.

Deliverables

Development of reliable methods for the quantitative, high-throughput assay of hundreds of ncAA-harboring proteins
Understanding of the catalytic mechanisms governing proton transfer at carbon in alanine racemase
Collection of over 10,000 gold-standard biochemical measurements of ncAA-containing proteins

Technical requirements

High-throughput microfluidic enzyme kinetics (HT-MEK) platform
Noncanonical amino acid (ncAA) site-specific incorporation into proteins
Parallel expression, purification, and quantitative assay of protein variants
Study of pyridoxal 5’-phosphate (PLP)-dependent enzymes, specifically alanine racemase
Systematic perturbation of electrostatic properties in enzyme active sites

Market context

inferred from NAICS

Professional, Scientific & Technical Services

NAICS 541714

US market size: $2.0T
Typical award: $25K – $50M

Typical buyers

All federal civilianDoDStates

Commonly required

8(a)WOSBSDVOSBPE/PMP

Sector-level estimate — full code lookup not yet in catalog.