{"doi":"10.1016/j.jbc.2025.111099","title":"Chaperone-assisted expression and purification of the AML-associated Src-family kinase Fgr in Escherichia coli","abstract":"Fgr, a member of the Src family of non-receptor tyrosine kinases, is expressed in myeloid hematopoietic cells and is a validated drug target in acute myeloid leukemia. Like other Src family kinases, Fgr consists of an N-terminal unique domain, SH3 and SH2 regulatory domains, a catalytic kinase domain, and a C-terminal tail. Notably, Fgr is constitutively active when ectopically expressed in fibroblasts, with kinase activity uncoupled from SH3-SH2 domain regulation, a feature that distinguishes it from other family members. To support structural and biochemical studies, we developed a chaperone-assisted method to express and purify soluble, active Fgr in E. coli . Fgr was co-expressed with C-terminal Src kinase to maintain C-terminal tail phosphorylation and with the protein-tyrosine phosphatase 1B catalytic domain to keep the activation loop dephosphorylated. While this strategy stabilizes soluble Src and Hck in E. coli , it was insufficient for Fgr. To enhance solubility, we added co-expression with the E. coli GroEL/ES chaperone complex. This significantly improved Fgr solubility, although the kinase initially remained bound to GroEL/ES. Washing column-bound GroEL/ES/Fgr with ATP-MgCl 2 partially dissociated the chaperone complex, enabling isolation of pure Fgr through subsequent size-exclusion and ion exchange chromatography. This method reliably produced highly pure, active recombinant Fgr, with consistent yields of 1 mg per 2 liters of bacterial culture.","journal":"Journal of Biological Chemistry","year":2025,"id":529612,"datarank":0.16479184330021646,"base_score":1.0986122886681096,"endowment":1.0986122886681096,"self_citation_contribution":0.16479184330021646,"citation_network_contribution":0.0,"self_endowment_contribution":0.16479184330021646,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9517,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":500792,"name":"Thomas E. Smithgall","orcid":"0000-0001-5238-3806","position":1,"is_corresponding":false},{"id":1308608,"name":"Giancarlo Gonzalez-Areizaga","orcid":null,"position":0,"is_corresponding":true}],"reference_count":29,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:50:56.971987Z","pmid":"41443422","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":[]}