Engineering Functional Membrane-Membrane Interfaces by InterSpy.
cell-free expression
membrane protein reconstitution
membrane-membrane interfaces
split-protein reconstitution
synthetic biology
Journal
Small (Weinheim an der Bergstrasse, Germany)
ISSN: 1613-6829
Titre abrégé: Small
Pays: Germany
ID NLM: 101235338
Informations de publication
Date de publication:
03 2023
03 2023
Historique:
revised:
26
04
2022
received:
04
04
2022
medline:
30
3
2023
pubmed:
27
5
2022
entrez:
26
5
2022
Statut:
ppublish
Résumé
Engineering synthetic interfaces between membranes has potential applications in designing non-native cellular communication pathways and creating synthetic tissues. Here, InterSpy is introduced as a synthetic biology tool consisting of a heterodimeric protein engineered to form and maintain membrane-membrane interfaces between apposing synthetic as well as cell membranes through the SpyTag/SpyCatcher interaction. The inclusion of split fluorescent protein fragments in InterSpy allows tracking of the formation of a membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. First, InterSpy is demonstrated by testing split protein designs using a mammalian cell-free expression (CFE) system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with the desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, InterSpy is demonstrated in mammalian cells by detecting fluorescence reconstitution of split protein at the membrane-membrane interface between two cells each expressing a component of InterSpy. InterSpy demonstrates the power of CFE systems in the functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful where cell-cell contacts and communication are recreated in a controlled manner using minimal components.
Identifiants
pubmed: 35618485
doi: 10.1002/smll.202202104
pmc: PMC9789529
mid: NIHMS1856434
doi:
Substances chimiques
Lipid Bilayers
0
Liposomes
0
Coloring Agents
0
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.
Langues
eng
Sous-ensembles de citation
IM
Pagination
e2202104Subventions
Organisme : NIBIB NIH HHS
ID : R01 EB030031
Pays : United States
Informations de copyright
© 2022 The Authors. Small published by Wiley-VCH GmbH.
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