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Expression of the human multidrug transporter in insect cells by a recombinant baculovirus
Authors:
Germann, U.A.
Willingham, M.C.
Pastan, I.
Gottesman, M.M. (National Institutes of Health, Bethesda, MD (USA))
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| Abstract: The plasma membrane associated human multidrug resistance (MDR1) gene product, known as the 170-kDa P-glycoprotein or the multidrug transporter, acts as an ATP-dependent efflux pump for various cytotoxic agents.^The authors expressed recombinant human multidrug transporter in a baculovirus expression system to obtain large quantities and further investigate its structure and mechanism of action.^MDR1 cDNA was inserted into the genome of the Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter.^Spodoptera frugiperda insect cells synthesized high levels of recombinant multidrug transporter 2-3 days after infection.^The transporter was localized by immunocytochemical methods on the external surface of the plasma membranes, in the Golgi apparatus, and within the nuclear envelope.^The human multidrug transporter expressed in insect cells is not susceptible to endoglycosidase F treatment and has a lower apparent molecular weight of 140,000, corresponding to the nonglycosylated precursor of its authentic counterpart expressed in multidrug-resistant cells.^Labeling experiments showed that the recombinant multidrug transporter is phosphorylated and can be photoaffinity labeled by ( 3H)azidopine, presumably at the same two sites as the native protein.^Various drugs and reversing agents compete with the ( 3H)azidopine binding reaction when added in excess, indicating that the recombinant human multidrug transporter expressed in insect cells is functionally similar to its authentic counterpart. |
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| Publication Date: |
06 Mar 1990
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| Resource Type: |
Journal Article
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| Resource Relation: |
Biochemistry ; Vol/Issue: 29:9
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| Country of Publication: |
United States
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| Language: |
English
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