Surface Display of Complex Enzymes by in Situ SpyCatcher-SpyTag Interaction.
Lpp-OmpA
biocatalysis
membrane proteins
self-assembly
surface display
Journal
Chembiochem : a European journal of chemical biology
ISSN: 1439-7633
Titre abrégé: Chembiochem
Pays: Germany
ID NLM: 100937360
Informations de publication
Date de publication:
03 08 2020
03 08 2020
Historique:
received:
18
02
2020
revised:
17
03
2020
pubmed:
18
3
2020
medline:
29
6
2021
entrez:
18
3
2020
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32182402
doi: 10.1002/cbic.202000102
pmc: PMC7497234
doi:
Substances chimiques
Enzymes
0
Recombinant Fusion Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2126-2131Informations de copyright
© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
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