Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Norton-Baker, Brenna; Denton, Mackenzie C. R.; Murphy, Natasha P.; Fram, Benjamin; Lim, Samuel; Erickson, Erika; Gauthier, Nicholas P.; Beckham, Gregg T.

doi:10.1038/s41598-024-64938-0

Title: Enabling high-throughput enzyme discovery and engineering with a low-cost, robot-assisted pipeline

Journal Article · 24 June 2024 · Scientific Reports

DOI: https://doi.org/10.1038/s41598-024-64938-0 · OSTI ID:2377872

Norton-Baker, Brenna; Denton, Mackenzie C. R.; Murphy, Natasha P.; Fram, Benjamin; Lim, Samuel; Erickson, Erika; Gauthier, Nicholas P.; Beckham, Gregg T.

Abstract As genomic databases expand and artificial intelligence tools advance, there is a growing demand for efficient characterization of large numbers of proteins. To this end, here we describe a generalizable pipeline for high-throughput protein purification using small-scale expression in E. coli and an affordable liquid-handling robot. This low-cost platform enables the purification of 96 proteins in parallel with minimal waste and is scalable for processing hundreds of proteins weekly per user. We demonstrate the performance of this method with the expression and purification of the leading poly(ethylene terephthalate) hydrolases reported in the literature. Replicate experiments demonstrated reproducibility and enzyme purity and yields (up to 400 µg) sufficient for comprehensive analyses of both thermostability and activity, generating a standardized benchmark dataset for comparing these plastic-degrading enzymes. The cost-effectiveness and ease of implementation of this platform render it broadly applicable to diverse protein characterization challenges in the biological sciences.

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Sponsoring Organization:: USDOE

Grant/Contract Number:: AC36-08GO28308; SC0022024

OSTI ID:: 2377872

Journal Information:: Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 14; ISSN 2045-2322

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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