Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1
Name
gzaa019.pdf
Description
Published version
Size
1.22 MB
Format
Adobe PDF
Checksum (MD5)
4a7dfe95af700cf5a5a16e19f8854855
Author(s)
Herzog, PL
Borghi, E
Traxlmayr, MW
Obinger, C
Sikes, HD
Peterbauer, CK
Date Issued
2020
Journal
Protein Engineering, Design and Selection
Publisher
Oxford University Press (OUP)
Version
Final published version
Abstract
© 2020 The Author(s). Accurate yet efficient high-throughput screenings have emerged as essential technology for enzyme engineering via directed evolution. Modern high-throughput screening platforms for oxidoreductases are commonly assisted by technologies such as surface display and rely on emulsification techniques to facilitate single-cell analysis via fluorescence-activated cell sorting. Empowered by the dramatically increased throughput, the screening of significantly larger sequence spaces in acceptable time frames is achieved but usually comes at the cost of restricted applicability. In this work, we tackle this problem by utilizing roGFP2-Orp1 as a fluorescent one-component detection system for enzymatic H2O2 formation. We determined the kinetic parameters of the roGFP2-Orp1 reaction with H2O2 and established an efficient immobilization technique for the sensor on Saccharomyces cerevisiae cells employing the lectin Concanavalin A. This allowed to realize a peroxide-sensing shell on enzyme-displaying cells, a system that was successfully employed to screen for H2O2 formation of enzyme variants in a whole-cell setting.
MIT Department
Massachusetts Institute of Technology. Department of Chemical Engineering
Terms of Use
Creative Commons Attribution NonCommercial License 4.0
Persistent DSpace Link
DOI of Published Version
https://doi.org/10.1093/protein/gzaa019
