Use of recombinant P-glycoprotein fragments to produce antibodies to the multidrug transporter.
Multidrug-resistance of human cancer cells may result from expression of a 170,000 dalton multidrug efflux pump called P-glycoprotein. To identify this multidrug transporter, and to study its structure and function, we have generated polyclonal rabbit antibodies against the amino-terminal and carboxy-terminal halves of the molecule using recombinant protein fragments produced in Escherichia coli. Two recombinant P-glycoprotein fragments, representing amino acids 140-228 and 919-1280, were overproduced in Escherichia coli by an inducible T7 expression system, gel-purified and injected into rabbits. Both antisera specifically immunoprecipitate 3H-azidopine and 35S-methionine labeled P-glycoprotein from multidrug-resistant cells and detect P-glycoprotein on Western blots with high sensitivity. Because these antisera were raised against epitopes in the amino- and carboxy-terminal halves of P-glycoprotein, they should be useful as research tools to define the function of these two halves of the molecule.
Tanaka
S
S
Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Currier
S J
SJ
Bruggemann
E P
EP
Ueda
K
K
Germann
U A
UA
Pastan
I
I
Gottesman
M M
MM
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001386:Azides; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004095:Dihydropyridines; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000276:immunology*; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D010957:Plasmids; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000345:Affinity Labels; D000906:Antibodies; D000911:Antibodies, Monoclonal; D001386:Azides; D004095:Dihydropyridines; D008562:Membrane Glycoproteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C041130:azidopine; D008715:Methionine