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Surface Display of Complex Enzymes by in Situ SpyCatcher-SpyTag Interaction

Gallus, S.; Peschke, T.; Paulsen, M.; Burgahn, T.; Niemeyer, C. M.; Rabe, K. S.

Abstract:
The display of complex proteins on the surface of cells is of great importance for protein engineering and other fields of biotechnology. Herein, we describe a modular approach, in which the membrane anchor protein Lpp-OmpA and a protein of interest (passenger) are expressed independently as genetically fused SpyCatcher and SpyTag units and assembled in situ by post-translational coupling. Using fluorescent proteins, we first demonstrate that this strategy allows the construct to be installed on the surface of E. coli cells. The scope of our approach was then demonstrated by using three different functional enzymes, the stereoselective ketoreductase Gre2p, the homotetrameric glucose 1-dehydrogenase GDH, and the bulky heme- and diflavin-containing cytochrome P450 BM3 (BM3). In all cases, the SpyCatcher-SpyTag method enabled the generation of functional whole-cell biocatalysts, even for the bulky BM3, which could not be displayed by conventional fusion with Lpp-OmpA. Furthermore, by using a GDH variant carrying an internal SpyTag, the system could be used to display an enzyme with unmodified N- and C-termini.

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Originalveröffentlichung
DOI: 10.1002/cbic.202000102
Scopus
Zitationen: 1
Web of Science
Zitationen: 1
Zugehörige Institution(en) am KIT Institut für Biologische Grenzflächen (IBG)
Publikationstyp Zeitschriftenaufsatz
Publikationsjahr 2020
Sprache Englisch
Identifikator ISSN: 1439-4227, 1439-7633
KITopen-ID: 1000119493
HGF-Programm 47.02.01 (POF III, LK 01) Zellpopul.auf Biofunk.Oberflächen IBG-1
Erschienen in ChemBioChem
Verlag Wiley-VCH Verlag
Vorab online veröffentlicht am 17.03.2020
Nachgewiesen in Scopus
Web of Science
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