{"doi":"10.1101/2021.10.22.465468","title":"PermaPhos <sup>Ser</sup> : autonomous synthesis of functional, permanently phosphorylated proteins","abstract":"ABSTRACT Installing stable, functional mimics of phosphorylated amino acids into proteins offers a powerful strategy to study protein regulation. Previously, a genetic code expansion (GCE) system was developed to translationally install non-hydrolyzable phosphoserine (nhpSer), with the γ-oxygen replaced with carbon, but it has seen limited usage. Here, we achieve a 40-fold improvement in this system by engineering into Escherichia coli a biosynthetic pathway that produces nhpSer from the central metabolite phosphoenolpyruvate. Using this “PermaPhos Ser ” system – an autonomous 21-amino acid E. coli expression system for incorporating nhpSer into target proteins – we show that nhpSer faithfully mimics the effects of phosphoserine in three stringent test cases: promoting 14-3-3/client complexation, disrupting 14-3-3 dimers, and activating GSK3β phosphorylation of the SARS-CoV-2 nucleocapsid protein. This facile access to nhpSer containing proteins should allow nhpSer to replace Asp and Glu as the go-to pSer phosphomimetic for proteins produced in E. coli .","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2021,"id":217971,"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":3,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9531,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":405023,"name":"Rachel Franklin","orcid":"0000-0002-8940-6204","position":1,"is_corresponding":false},{"id":815798,"name":"Amber Vogel","orcid":null,"position":2,"is_corresponding":false},{"id":815799,"name":"Stanislau Stanisheuski","orcid":null,"position":3,"is_corresponding":false},{"id":289459,"name":"Patrick N. Reardon","orcid":"0000-0002-6858-0086","position":4,"is_corresponding":false},{"id":815313,"name":"Nikolai N. Sluchanko","orcid":"0000-0002-8608-1416","position":5,"is_corresponding":false},{"id":14604,"name":"Joseph S. Beckman","orcid":"0000-0003-1855-4653","position":6,"is_corresponding":false},{"id":475808,"name":"P. Andrew Karplus","orcid":"0000-0001-8725-6292","position":7,"is_corresponding":false},{"id":395007,"name":"Ryan A. Mehl","orcid":"0000-0003-2932-4941","position":8,"is_corresponding":false},{"id":395005,"name":"Richard B. Cooley","orcid":"0000-0003-3928-2757","position":9,"is_corresponding":false},{"id":475806,"name":"Phillip Zhu","orcid":"0000-0002-9756-2637","position":0,"is_corresponding":true}],"reference_count":70,"raw_metadata":null,"created_at":"2026-07-18T23:53:24.683898Z","pmid":"34931187","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":[]}