Developing a cell-bound detection system for the screening of oxidase activity using the fluorescent peroxide sensor roGFP2-Orp1
| Autor: | Michael W. Traxlmayr, E Borghi, Christian Obinger, Peter L. Herzog, Hadley D. Sikes, Clemens K. Peterbauer |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Saccharomyces cerevisiae Proteins
H2O2 Recombinant Fusion Proteins Saccharomyces cerevisiae Cell Green Fluorescent Proteins Bioengineering 01 natural sciences Biochemistry 03 medical and health sciences medicine Molecular Biology 030304 developmental biology AcademicSubjects/SCI01030 0303 health sciences Oxidase test biology 010405 organic chemistry Chemistry flow cytometry screening Protein engineering Hydrogen Peroxide Cell sorting biology.organism_classification Directed evolution Fluorescence 0104 chemical sciences enzyme surface display medicine.anatomical_structure Concanavalin A biology.protein Original Article Biological system Oxidoreductases Biotechnology |
| Zdroj: | Protein Engineering, Design and Selection |
| ISSN: | 1741-0134 1741-0126 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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