{"doi":"10.17504/protocols.io.e6nvwqw6dvmk/v1","title":"Large scale MPro MERS-CoV protein 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 novel single-use Bubble Column Reactor (suBCR) array system for large-scale recombinant protein production in E. coli, specifically demonstrated with MERS-CoV main protease (MPro). Traditional shake-flask methods are inadequate for meeting the high protein quantities required for structure-based drug discovery (SBDD) workflows, particularly when multiple protein constructs need to be evaluated simultaneously. The suBCR system enables parallel 1-liter E. coli batch cultivation using disposable bioreactor bags arranged in a heated water bath with controlled aeration. The protocol utilizes auto-induction Terrific Broth media supplemented with antifoam, antibiotics, and glycerol. Cultures are inoculated with starter cultures and grown at 37°C for approximately 4 hours until reaching exponential phase, followed by overnight protein expression at ambient temperature (25-27°C) for 16-20 hours. The system achieved successful expression of MERS-CoV MPro protein with high yields, producing 199.5 g wet cell weight from 6 liters total culture volume (OD₆₀₀ = 39.6). Quality control using rapid nickel-magnetic bead purification and SDS-PAGE analysis confirmed successful protein overexpression. The suBCR array addresses the bottleneck of insufficient protein quantities in structural biology pipelines while reducing labor burden and increasing throughput compared to conventional methods. This scalable, cost-effective approach represents a significant advancement in protein production methodology for structural biology and drug discovery applications, offering a practical solution for laboratories requiring multiple protein constructs for crystallographic studies.","journal":null,"year":2025,"id":568778,"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":0.9576,"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":{"citation_network_status":"fetched"},"created_at":"2026-07-19T02:56:55.795846Z","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":[]}