title: Efficient production of secreted staphylococcal antigens in a non-lysing and proteolytically reduced Lactococcus lactis strain.
abstract: Cell surface-exposed and secreted proteins are attractive targets for vaccination against pathogenic gram-positive bacteria. To obtain sufficient amounts of such antigens, efficient protein production platforms are needed. In this study, a pipeline for the production and purification of surface-exposed and secreted antigens of the gram-positive bacterial pathogen Staphylococcus aureus is presented. Cytoplasmic or extracellular production of S. aureus antigens was achieved using the Lactococcus lactis strain PA1001, which lacks the major extracellular protease HtrA and the autolysin AcmA to minimize proteolysis and cell lysis, respectively. For most tested S. aureus antigens, secretory production directed by the signal peptide of the major secreted protein Usp45 of L. lactis resulted in higher yields than intracellular production without a signal peptide. Additionally, secretory production of His-tagged antigens allowed their facile one-step purification from the growth medium by metal affinity chromatography. For three of the purified antigens, biological activity was confirmed through enzyme activity assays. We, furthermore, show that the present pipeline can be used to produce staphylococcal antigens with an N-terminal AVI-tag for site-specific labeling with biotin or a C-terminal cell wall-binding domain for cell surface display. We conclude that our L. lactis-based pipeline allows the efficient production of S. aureus antigens and their subsequent purification in one step.
authors.affiliation: Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, PO box 30001, 9700 RB, Groningen, The Netherlands.
authors.forename: Jolanda
authors.initials: J
authors.lastname: Neef
authors.forename: Danny G A M
authors.initials: DG
authors.lastname: Koedijk
authors.forename: Tjibbe
authors.initials: T
authors.lastname: Bosma
authors.forename: Jan Maarten
authors.initials: JM
authors.lastname: van Dijl
authors.forename: Girbe
authors.initials: G
authors.lastname: Buist
mesh_terms: D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001433:Bacteriolysis; D002846:Chromatography, Affinity; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013211:Staphylococcus aureus / Q000235:genetics*
chemical_list: D000942:Antigens, Bacterial; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins