{"doi":"10.17504/protocols.io.14egnypbqv5d/v1","title":"Large scale EV-A71 2A protease mature and precursor expression setup and operation of Single Use Bubble Column Reactors: Litre-Scale Expression of Recombinant Proteins for Structural Biology and Drug Design (SBDD) v1","abstract":"This protocol describes a single-use Bubble Column Reactor (suBCR) system for large-scale recombinant protein production in E. coli, addressing limitations of traditional shake-flask methods for structure-based drug discovery. The system enables parallel 1-liter batch cultivation using disposable bioreactor bags in a heated water bath with controlled aeration. Two enterovirus 2A protease variants were successfully expressed: wild-type Human Coxsackievirus A16 2A protease (Addgene 228632) and an inactive Coxsackievirus A71 2A protease with C110A mutation (Addgene 228633).The C110A mutation preserves the VP1-2A junction while eliminating catalytic activity, providing active and inactive variants for comparative studies that are suitable for crystallography. Cultures are grown at 37°C for 4 hours until exponential phase, then expressed overnight at ambient temperature (25-27°C) using auto-induction Terrific Broth media. The system achieved high yields with culture volumes of 5-22.6 L producing wet cell weights of 163.2-225.6 g. Quality control via nickel-magnetic bead purification and SDS-PAGE confirmed successful overexpression of both constructs. This scalable, cost-effective approach significantly advances protein production methodology for structural biology and drug discovery, offering increased throughput and reduced labor compared to conventional methods while providing sufficient protein quantities for crystallographic studies and antiviral drug development.","journal":null,"year":2025,"id":569774,"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":0.9568,"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":1090393,"name":"Nathan T. Wright","orcid":"0000-0003-0177-6129","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:57:03.510013Z","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":[]}