{"doi":"10.1016/j.jbc.2023.105031","title":"Expression conditions and characterization of a novelly constructed lipoprotein intended as a vaccine to prevent human Haemophilus influenzae infections","abstract":"Bacterial lipoproteins are structurally divided into two groups, based on their lipid moieties: diacylated (present in Gram-positive bacteria) and triacylated (present in some Gram-positive and most Gram-negative bacteria). Diacylated and triacylated lipid moieties differ by a single amide-linked fatty acid chain. Lipoproteins induce host innate immune responses by the mammalian Toll-like receptor 2 (TLR2). In this study, we added a lipid moiety to recombinant OMP26, a native nonlipidated (NL) membrane protein of Haemophilus influenzae, and characterized it extensively under different expression conditions using flow cytometry, LC/MS, and MALDI-TOF. We also investigated the ability of NL and lipidated (L) OMP26 to induce in vitro stimulation of HEK Blue-hTLR2-TR1 and hTLR-TLR6 cells. Our L-OMP26 was predominantly expressed in diacylated form, so we employed an additional gene copy of apolipoprotein N-acetyltransferase enzyme (Lnt)-rich Escherichia coli strain that further acylates the diacyl lipoproteins to enhance the production of triacylated L-OMP26. The diacyl and triacyl versions of L-OMP26, intended as a vaccine for use in humans, were characterized and evaluated as protein vaccine components in a mouse model. We found that the diacyl and triacyl L-OMP26 protein formulations differed markedly in their immune-stimulatory activity, with diacylated L-OMP26 stimulating higher adaptive immune responses compared with triacylated L-OMP26 and both stimulating higher adaptive immune response compared to NL-OMP26. We also constructed and characterized an L-OMP26φNL-P6 fusion protein, where NL-P6 protein (a commonly studied H. influenzae vaccine candidate) was recombinantly fused to L-OMP26. We observed a similar pattern of lipidation (predominantly diacylated) in the L-OMP26φNL-P6 fusion protein.","journal":"Journal of Biological Chemistry","year":2023,"id":364201,"datarank":0.31191623125197543,"base_score":2.0794415416798357,"endowment":2.0794415416798357,"self_citation_contribution":0.31191623125197543,"citation_network_contribution":0.0,"self_endowment_contribution":0.31191623125197543,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":7,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9487,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1119434,"name":"Jill Mangiafesto","orcid":null,"position":1,"is_corresponding":false},{"id":1119435,"name":"Karin Pryharski","orcid":null,"position":2,"is_corresponding":false},{"id":364193,"name":"Sailee Rasam","orcid":"0000-0001-8051-8034","position":3,"is_corresponding":false},{"id":1119436,"name":"Robert J. Zagursky","orcid":null,"position":4,"is_corresponding":false},{"id":298676,"name":"Michael E. Pichichero","orcid":"0000-0002-8276-3938","position":5,"is_corresponding":false},{"id":298673,"name":"Ravinder Kaur","orcid":"0000-0001-6539-5339","position":0,"is_corresponding":true}],"reference_count":41,"raw_metadata":{"citation_network_status":"fetched"},"created_at":"2026-07-19T01:14:36.728255Z","pmid":"37437888","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":[]}