High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution
Abstract
Advances in directed evolution have led to an exploration of new and important chemical transformations; however, many of these efforts still rely on the use of low-throughput chromatography-based screening methods. We present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for C–H alkylation. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed.
Additional Information
© 2020 American Chemical Society. Received: July 20, 2020; Published: November 11, 2020. We thank Dr. Ruijie Zhang for initial discussions and Dr. Sabine-Brinkmann-Chen for critical review of the manuscript. A.J.P. and D.J.W. are supported by the National Science Foundation Graduate Research Fellowship under Grant Nos. DGE-1842165 and DGE-174530. Research reported in this publication was supported by the Department of Defense, Defense Threat Reduction Agency, under award HDTRA1-15-1-0052 (to M.M.) and by the US Army Research Office Institute for Collaborative Biotechnologies cooperative agreement W911NF-19-2-0026 (to F.H.A. and X.H.). Author Contributions: A.J.P. and D.J.W. contributed equally to this work. The authors declare no competing financial interest.Attached Files
Supplemental Material - ja0c07828_si_001.pdf
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Additional details
- Eprint ID
- 106617
- Resolver ID
- CaltechAUTHORS:20201111-125109727
- DGE-1842165
- NSF Graduate Research Fellowship
- DGE-174530
- NSF Graduate Research Fellowship
- HDTRA1-15-1-0052
- Defense Threat Reduction Agency (DTRA)
- W911NF-19-2-0026
- Army Research Office (ARO)
- Created
-
2020-11-11Created from EPrint's datestamp field
- Updated
-
2021-11-16Created from EPrint's last_modified field
- Caltech groups
- Division of Biology and Biological Engineering