title: An efficient depyrogenation method for recombinant bacterial outer membrane lipoproteins.
abstract: Bacterial outer membrane lipoproteins are anchored in the outer membrane lipid layer in close association with lipopolysaccharides (LPS) and with other hydrophobic membrane proteins, making their purification technically challenging. We have previously shown that a thorough delipidation of outer membrane preparations from the Escherichia coli expression host is an important step to eliminate contaminant proteins when purifying recombinant antigens expressed in fusion with the Pseudomonas aeruginosa OprI lipoprotein. Here we report the cloning and expression of three antigens in fusion with OprI (ovalbumin, eGFP and BbPDI) and our efforts to deal with the variable LPS contamination levels observed in different batches of purified lipoproteins. The use of polymyxin B columns or endotoxin removal polycationic magnetic beads for depyrogenation of purified lipoproteins resulted in high protein losses and the use of Triton X-114 or sodium deoxycholate during the course of affinity chromatography showed to be ineffective to reduce LPS contamination. Instead, performing a hot phenol/water LPS extraction from outer membrane preparations prior to metal affinity chromatography allowed the purification of the recombinant fusion lipoproteins with LPS contents below 0.02EU/μg of protein. The purified recombinant lipoproteins retain their capacity to stimulate bone marrow-derived dendritic cells allowing for the study of their immunomodulatory properties through TLR2/1. This is a simple and easy to scale up method that can also be considered for the purification of other outer membrane lipoproteins.
authors.affiliation: Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
authors.forename: Afonso P
authors.initials: AP
authors.lastname: Basto
authors.affiliation: Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; Instituto de Investigação Científica Tropical, CVZ, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
authors.forename: Joana
authors.initials: J
authors.lastname: Morais
authors.affiliation: Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; Instituto de Investigação Científica Tropical, CVZ, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal.
authors.forename: Eduardo
authors.initials: E
authors.lastname: Marcelino
authors.affiliation: Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; Instituto de Investigação Científica Tropical, CVZ, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal. Electronic address: alexandre@fmv.ulisboa.pt.
authors.forename: Alexandre
authors.initials: A
authors.lastname: Leitão
authors.affiliation: Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal; Instituto de Investigação Científica Tropical, CVZ, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal. Electronic address: dulcesantos@fmv.ulisboa.pt.
authors.forename: Dulce M
authors.initials: DM
authors.lastname: Santos
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D005591:Chemical Fractionation / Q000379:methods*; D002645:Chickens; D002846:Chromatography, Affinity; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology; D004926:Escherichia coli; D051379:Mice; D010047:Ovalbumin / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology
chemical_list: D001425:Bacterial Outer Membrane Proteins; D011993:Recombinant Fusion Proteins; D010047:Ovalbumin
keywords: Bacterial outer membrane proteins; Endotoxin; Lipopolysaccharide; Outer membrane lipoprotein I; Phenol; TLR