A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

• Published in: Journal of biotechnology Vol. 157; no. 1; pp. 50 - 63
• Main Authors: Basto, Afonso P., Piedade, João, Ramalho, Ruben, Alves, Susana, Soares, Helena, Cornelis, Pierre, Martins, Carlos, Leitão, Alexandre
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.01.2012; Elsevier
• Subjects: adaptive immunity; Adjuvant; affinity chromatography; African swine fever virus; African Swine Fever Virus - genetics; Animals; antigens; Antigens, Viral - genetics; bacteria; Bacterial outer membrane proteins; Bacterial Proteins - biosynthesis; Bacterial Proteins - genetics; Bacterial Proteins - immunology; Base Sequence; Biological and medical sciences; Biotechnology; Cells, Cultured; Cloning vector; Cloning, Molecular - methods; Dendritic Cells - immunology; Escherichia coli; Escherichia coli - genetics; Escherichia coli - metabolism; Fundamental and applied biological sciences. Psychology; Genetic Vectors - genetics; Immunomodulation; lipoproteins; Lipoproteins - biosynthesis; Lipoproteins - genetics; Lipoproteins - immunology; Macrophages - immunology; Mice; Molecular Sequence Data; Orf virus; Outer membrane lipoprotein I; palmitic acid; Pseudomonas; Pseudomonas aeruginosa; Pseudomonas aeruginosa - genetics; receptors; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Tumor Necrosis Factor-alpha - metabolism; vaccines; ELFA; DM; MHC; DDM; Pseudomonas; PRR; OG; Adjuvant; TLR; ASFV; OM; Immunomodulation; MS; OMF; Bacterial outer membrane proteins; TCA; PMSF; Cloning vector; ALFA; IPTG; OMV; CTL; FA; MCS; OprI; GLC; Outer membrane lipoprotein I; Pseudomonadales; Lipoprotein; Recombinant cell; External membrane; Cell wall; Protein; Formulation; Production; Pseudomonadaceae; Bacteria; Molecular cloning; Vector
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2011.11.006

► We propose a new system for expression of antigens fused with the OprI lipoprotein. ► It offers tight control of expression and metal affinity chromatography purification. ► The OprI expressed by the system is triacylated, mainly by palmitic acid residues. ► The cloned antigens can be obtained in three bacterial cell wall-derived formulations. ► Applications include studies on immunization and on modulation of acquired immunity. The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.