{"doi":"10.1021/jacs.2c10194","title":"Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases","abstract":"Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial processes (e.g., the production of antibody–drug conjugates and immobilization of enzymes for biocatalysis). A popular approach involves reacting a free N-terminal cysteine with a variety of electrophilic reagents. However, current methods for generating proteins with N-terminal cysteines have significant limitations. Herein we report a novel, efficient, and convenient method for producing recombinant proteins with free N-terminal cysteines by genetically fusing a Met-Pro-Cys sequence to the N-terminus of a protein of interest and subjecting the recombinant protein to the sequential action of methionine and proline aminopeptidases. The resulting protein was site-specifically labeled at the N-terminus with fluorescein and a cyclic cell-penetrating peptide through native chemical ligation and a 2-cyanobenzothiazole moiety, respectively. In addition, the optimal recognition sequence of Aeromonas sobria proline aminopeptidase was determined by screening a combinatorial peptide library and incorporated into the N-terminus of a protein of interest for most efficient N-terminal processing.","journal":"Journal of the American Chemical Society","year":2022,"id":272220,"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":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9415,"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":938206,"name":"Emily R. Sekera","orcid":"0000-0002-1668-3227","position":1,"is_corresponding":false},{"id":263188,"name":"Amar Sarkar","orcid":"0000-0002-6114-1873","position":2,"is_corresponding":false},{"id":37627,"name":"Amanda B. Hummon","orcid":"0000-0002-1969-9013","position":3,"is_corresponding":false},{"id":159781,"name":"Dehua Pei","orcid":"0000-0002-2057-6934","position":4,"is_corresponding":false},{"id":938755,"name":"Jordan P Hempfling","orcid":null,"position":0,"is_corresponding":true}],"reference_count":32,"raw_metadata":null,"created_at":"2026-07-19T00:27:43.806977Z","pmid":"36378906","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":[]}