A-protein from achromogenic atypical Aeromonas salmonicida: molecular cloning, expression, purification, and characterization.
Achromogenic atypical Aeromonas salmonicida is the causative agent of goldfish ulcer disease. Virulence of this bacterium is associated with the production of a paracrystalline outer membrane A-layer protein. The species-specific structural gene for the monomeric form of A-protein was cloned into a pET-3d plasmid in order to express and produce a recombinant form of the protein in Escherichia coli BL21(DE3). The induced protein was isolated from inclusion bodies by a simple solubilization-renaturation procedure and purified by ion exchange chromatography on Q-Sepharose to over 95% pure monomeric protein. Recombinant A-protein was compared by biochemical, immunological, and molecular methods with the A-protein isolated from atypical A. salmonicida bacterial cells by the glycine and the membrane extraction methods. The recombinant form was found to be undistinguishable from the wild type when examined by SDS-PAGE and gel filtration chromatography. The immunological similarity of the protein samples was demonstrated by employing polyclonal and monoclonal antibodies in ELISA and Western blot techniques. All forms of A-protein were found to activate the secretion of tumor necrosis factor alpha from murine macrophage. To date, this represents the first large-scale production of biologically active recombinant A-protein.
Maurice
S
S
Institute of Biochemistry, Food Science and Nutrition, Faculty of Agriculture, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rechovot, Israel.
Hädge
D
D
Dekel
M
M
Friedman
A
A
Gertler
A
A
Shoseyov
O
O
D000333:Aeromonas / Q000737:chemistry*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry; D002478:Cells, Cultured; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003601:Cytotoxicity Tests, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D017737:Macrophages, Peritoneal / Q000737:chemistry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013997:Time Factors; D037521:Virulence Factors
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D037521:Virulence Factors