{"doi":"10.1101/2022.04.04.487023","title":"Engineering functional membrane-membrane interface by InterSpy","abstract":"Abstract 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 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2022,"id":306872,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9515,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2022-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":724802,"name":"Chintan Patel","orcid":"0000-0001-6245-2406","position":1,"is_corresponding":false},{"id":874923,"name":"Sonisilpa Mohapatra","orcid":"0000-0002-7839-8122","position":2,"is_corresponding":false},{"id":1001205,"name":"Sung‐Won Hwang","orcid":"0000-0003-1686-7090","position":3,"is_corresponding":false},{"id":48090,"name":"Tunc Kayikcioglu","orcid":"0000-0003-2205-8062","position":4,"is_corresponding":false},{"id":661680,"name":"Yashar Bashirzadeh","orcid":"0000-0003-0688-1454","position":5,"is_corresponding":false},{"id":356962,"name":"Allen P. Liu","orcid":"0000-0002-0309-7018","position":6,"is_corresponding":false},{"id":22168,"name":"Taekjip Ha","orcid":"0000-0003-2195-6258","position":7,"is_corresponding":false},{"id":470990,"name":"Hossein Moghimianavval","orcid":"0000-0003-3358-0630","position":0,"is_corresponding":true}],"reference_count":73,"raw_metadata":null,"created_at":"2026-07-19T00:32:52.703737Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}