title: Enhanced expression of codon optimized Mycobacterium avium subsp. paratuberculosis antigens in Lactobacillus salivarius.
abstract: It is well documented that open reading frames containing high GC content show poor expression in A+T rich hosts. Specifically, G+C-rich codon usage is a limiting factor in heterologous expression of Mycobacterium avium subsp. paratuberculosis (MAP) proteins using Lactobacillus salivarius. However, re-engineering opening reading frames through synonymous substitutions can offset codon bias and greatly enhance MAP protein production in this host. In this report, we demonstrate that codon-usage manipulation of MAP2121c can enhance the heterologous expression of the major membrane protein (MMP), analogous to the form in which it is produced natively by MAP bacilli. When heterologously over-expressed, antigenic determinants were preserved in synthetic MMP proteins as shown by monoclonal antibody mediated ELISA. Moreover, MMP is a membrane protein in MAP, which is also targeted to the cellular surface of recombinant L. salivarius at levels comparable to MAP. Additionally, we previously engineered MAP3733c (encoding MptD) and show herein that MptD displays the tendency to associate with the cytoplasmic membrane boundary under confocal microscopy and the intracellularly accumulated protein selectively adheres to the MptD-specific bacteriophage fMptD. This work demonstrates there is potential for L. salivarius as a viable antigen delivery vehicle for MAP, which may provide an effective mucosal vaccine against Johne's disease.
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Johnston
authors.affiliation: United States Department of Agriculture - Agricultural Research Service, National Animal Disease Center Ames, IA, USA.
authors.forename: John P
authors.initials: JP
authors.lastname: Bannantine
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Rodney
authors.initials: R
authors.lastname: Govender
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Lorraine
authors.initials: L
authors.lastname: Endersen
authors.affiliation: School of Engineering and Science, Jacobs University Bremen Bremen, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Pletzer
authors.affiliation: School of Engineering and Science, Jacobs University Bremen Bremen, Germany.
authors.forename: Helge
authors.initials: H
authors.lastname: Weingart
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Aidan
authors.initials: A
authors.lastname: Coffey
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Jim
authors.initials: J
authors.lastname: O'Mahony
authors.affiliation: Biological Sciences Department, Cork Institute of Technology Cork, Ireland.
authors.forename: Roy D
authors.initials: RD
authors.lastname: Sleator
mesh_terms: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D003062:Codon; D003593:Cytoplasm / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D007778:Lactobacillus / Q000235:genetics*; D016927:Mycobacterium avium subsp. paratuberculosis / Q000235:genetics* / Q000276:immunology; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D003062:Codon; D000939:Epitopes; D011993:Recombinant Fusion Proteins
keywords: Johne's disease; MAP antigens; MAP vaccine; MMP; MptD; codon optimization; expression host; paratuberculosis