Improved purification of native meningococcal porin PorB and studies on its structure/function.
The outer membrane protein PorB of Neisseria meningitidis is a pore-forming protein which has various effects on eukaryotic cells. It has been shown to (1) up-regulate the surface expression of the co-stimulatory molecule CD86 and of MHC class II (which are TLR2/MyD88 dependent and related to the porin's immune-potentiating ability), (2) be involved in prevention of apoptosis by modulating the mitochondrial membrane potential, and (3) form pores in eukaryotic cells. As an outer membrane protein, its native trimeric form isolation is complicated by its insoluble nature, requiring the presence of detergent throughout the whole procedure, and by its tight association with other outer membrane components, such as neisserial LOS or lipoproteins. In this study, an improved chromatographic purification method to obtain an homogeneous product free of endotoxin and lipoprotein is described, without loss of any of the above-mentioned properties of the porin. Furthermore, we have investigated the requirement of the native trimeric structure for the porin's activity. Inactivation of functional PorB trimers into non-functional monomers was achieved by incubation on ice. Thus, routine long- and medium-term storage at low temperature may be a cause of porin inactivation.
Massari
Paola
P
Evans BioMedical Research Center, Division of Infectious Diseases, Department of Medicine, Boston University School of Medicine, Boston Medical Center, Boston, MA, USA.
King
Carol A
CA
MacLeod
Heather
H
Wetzler
Lee M
LM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001402:B-Lymphocytes / Q000187:drug effects; D051940:B7-2 Antigen / Q000378:metabolism; D011232:Chemical Precipitation; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007425:Intracellular Membranes / Q000187:drug effects / Q000502:physiology; D008213:Lymphocyte Activation / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D008928:Mitochondria / Q000187:drug effects; D008970:Molecular Weight; D018272:Porins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011489:Protein Denaturation; D019311:Staurosporine / Q000494:pharmacology
D051940:B7-2 Antigen; D018272:Porins; C057699:porin protein, Neisseria; D019311:Staurosporine