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Surface Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction
Author(s) -
Gallus Sabrina,
Peschke Theo,
Paulsen Malte,
Burgahn Teresa,
Niemeyer Christof M.,
Rabe Kersten S.
Publication year - 2020
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000102
Subject(s) - protein engineering , enzyme , biochemistry , chemistry , biology
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.