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Sci Rep. 2014 Dec 22;4:7570. doi: 10.1038/srep07570.

An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

Author information

1
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA.
2
Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA.
3
1] School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, NY 14853 USA [2] Department of Biomedical Engineering, Cornell University, Ithaca, NY 14853 USA.

Abstract

The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

PMID:
25531212
PMCID:
PMC4273604
DOI:
10.1038/srep07570
[Indexed for MEDLINE]
Free PMC Article

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