Raising antibodies against OprD, an outer membrane protein of Pseudomonas aeruginosa using translational fusions to MalE.
OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).
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Department of Genetics and Microbiology, Centre Médical Universitaire (CMU), 9, ave de Champel, CH-1211 4, Geneva, Switzerland.
Pechère
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J
Kok
M
M
D018528:ATP-Binding Cassette Transporters; D000688:Amylose / Q000378:metabolism; D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D019897:Periplasm / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D018272:Porins / Q000235:genetics* / Q000276:immunology; D011485:Protein Binding; D014176:Protein Biosynthesis; D011550:Pseudomonas aeruginosa / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C461619:MalE protein, E coli; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D018272:Porins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C077157:OprD protein, Pseudomonas aeruginosa; D008320:Maltose; D000688:Amylose