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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.
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Split Green Fluorescent Proteins: Scope, Limitations, and Outlook
Many proteins can be split into fragments that spontaneously reassemble, without covalent linkage, into a functional protein. For split green fluorescent proteins (GFPs), fragment reassembly leads to a fluorescent readout, which has been widely used to investigate protein–protein interactions. We review the scope and limitations of this approach as well as other diverse applications of split GFPs as versatile sensors, molecular glues, optogenetic tools, and platforms for photophysical studies.
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- Award ID(s):
- 1740645
- PAR ID:
- 10111949
- Date Published:
- Journal Name:
- Annual Review of Biophysics
- Volume:
- 48
- Issue:
- 1
- ISSN:
- 1936-122X
- Page Range / eLocation ID:
- 19 to 44
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1146/annurev-biophys-051013-022846
