Pseudomonas exotoxin fusion proteins are potent immunogens for raising antibodies against P-glycoprotein.
Antibodies to specific regions of human P-glycoprotein have been difficult to obtain. We developed a method to express in E. coli fusions between Pseudomonas exotoxin and specific regions of human P-glycoprotein. We used the polymerase chain reaction to amplify the desired regions of MDR1 cDNA and to introduce appropriate restriction sites. These fragments were cloned into the 3' end of the Pseudomonas exotoxin gene. With this system we produced large amounts of fusion proteins for immunizations, and we obtained positive rabbit antiserum against P-glycoprotein with most of these antigens. We now have a comprehensive panel of polyclonal antibodies against P-glycoprotein. This system should be generally useful to raise antibodies against other eukaryotic proteins that are difficult to prepare in large quantities.
Bruggemann
E P
EP
National Cancer Institute, National Institutes of Health.
Chaudhary
V
V
Gottesman
M M
MM
Pastan
I
I
D036002:ADP Ribose Transferases; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis* / Q000276:immunology; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics; D005098:Exotoxins / Q000235:genetics / Q000276:immunology*; D005798:Genes, Bacterial; D006801:Humans; D008562:Membrane Glycoproteins / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology*; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D004247:DNA; D005098:Exotoxins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A