Engineering functional membrane-membrane interface by InterSpy

Abstract

AbstractEngineering 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 SpyTag/SpyCatcher interaction. Inclusion of split fluorescent protein fragments in the designed InterSpy toolkit allows tracking the formation of membrane-membrane interface and reconstitution of functional fluorescent protein in the space between apposing membranes. We first demonstrate InterSpy by testing split protein designs using a mammalian cell-free expression system. By utilizing co-translational helix insertion, cell-free synthesized InterSpy fragments are incorporated into the membrane of liposomes and supported lipid bilayers with a desired topology. Functional reconstitution of split fluorescent protein between the membranes is strictly dependent on SpyTag/SpyCatcher. Finally, since InterSpy is fully genetically encoded, the engineered system is adapted to cells and showcased. InterSpy demonstrates the power of cell-free expression systems in functional reconstitution of synthetic membrane interfaces via proximity-inducing proteins. This technology may also prove useful for synthetic biology where cell-cell contacts and communication are recreated in a controlled manner using minimal components.