Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

Germann, Ursula A.; Willingham, Mark C.; Pastan, Ira; Gottesman, Michael M.

doi:10.1021/bi00461a013

Cited by 127 publications

(93 citation statements)

References 42 publications

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“…There are known to be variations in glycosylation of P-glycoproteins derived from different mouse tissues [21] but whether these reflect differences in functional activity is unclear. Though complete N-glycosylation of the human MDR1 gene product is not thought essential for drug binding [30] or transport, N-glycosylation may affect drug transport indirectly by influencing routing and stability of P-glycoprotein [311.…”

Section: Discussionmentioning

confidence: 99%

Comparisons of P‐glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta

1995

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In vivo expression of P-glycoprotein in isolated rat brain mierovessels is compared with that in vitro in primary cultures of brain endothelial cells. More P-glycoprotein is detected by Western immunoblotting in microvessels than in cultured endothelium. RT-PCR with isnform-specific primers and immunobiotting with a mdrlb-specific antibody reveals only mdrla in vivo but both mdrla and mdrlb in vitro. Thus mdrla decreases whereas mdrlb increases during culture. P-Glycoprotein activity is evident in vitro, with resistance modulators, e.g. verapamil, producing increases in intracenular [3H]vincristine accumulation. Endothelial cells cultured from epididymal fat pad microvaseulature and aorta contain little or no P-glycoprotein. Here, resistance modulators are less effective.

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Section: Discussionmentioning

confidence: 99%

Comparisons of P‐glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta

1995

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“…Other examples are the ABC transporters multidrug resistance P-glycoprotein (MDR) [36,37] and the cystic fibrosis transmembrane conductance regulator (CFTR) [38].…”

Section: Introductionmentioning

confidence: 99%

Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells

et al. 1994

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Using the baculovirus expression system the gene products of human tapl and tap2 were over-expressed as wild-type as well as oligohistidine fusion proteins in Spodopterafrugiperda (Sf9) insect cells, Both gene products were co-expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATE For the first time the functional expression ofthe human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.

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“…Specifically, the mechanism of P-gp action remains unknown and can only be solved by purification of P-gp to homogeneity, followed by reconstitution into lipid vesicles. Although recent attempts in this direction show promising results, they are hampered by the limited availability of starting material from either drugresistant cell lines (35) or insect cells overexpressing large amounts ofrecombinant protein (30,36). The yeast expression system described here should allow the rapid and simplified production of large amounts of biologically active P-gp, alleviating a key problem to purification and reconstitution.…”

Section: Resultsmentioning

confidence: 99%

Functional expression of P-glycoprotein encoded by the mouse mdr3 gene in yeast cells.

Ruetz

Raymond

Gros

1993

Proc. Natl. Acad. Sci. U.S.A.

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We have expressed P-glycoprotein (P-gp) encoded by the mouse mdr3 gene in the yeast Saccharomyces cerevisiae and have developed an experimental protocol to isolate and purify inside-out plasma membrane vesicles (IOVs) from these ceUls. Biochemical characterization of IOVs from control and P-gp-expressing cels isolated by this procedure show that they are greatly enriched for plasma membrane markers, are tightly sealed, and are competent for D-glucose transport. P-gp expression in these vesicles results in the appearance of a specific ATP-dependent and temperaturesensitive transport of the drugs colchicine and vinblastine that is osmotically sensitive. P-gp-mediated drug transport into these IOVs is inhibited by a known P-gp modulator, verapamil, and can be abrogated by prior incubation of the IOVs with an anti-P-gp antibody. A Ser-939 --Phe mutation within the predicted transmembrane domain 11 of P-gp, which is known to modulate its function in mammalian cells, drastically reduces drug transport in IOVs obtained from yeast cells expressing the mutant protein. The successful demonstration of active drug transport into IOVs from P-gp-expressing yeast cells indicates that P-gp can mediate both chemotherapeutic drugs and a-pheromone transport in yeast cells.Multidrug resistance (MDR) is caused in vitro and in vivo by overexpression of a membrane phosphoglycoprotein, P-glycoprotein (P-gp) (1). P-gp is encoded by a small family of related mdr genes composed of three members in rodents (mdrl, mdr2, and mdr3) (2-4) and two in humans (MDR] and MDR2; now designated PGY1 and PGY2) (5, 6). The prototype P-gp is formed by two homologous halves, each composed of six predicted transmembrane domains and one nucleotide-binding (NB) fold (7). The P-gp family is also part of a very large family of ATP-binding cassette membrane transporters (8) with members in prokaryotes and lower and higher eukaryotes, including the yeast Saccharomyces cerevisiae a peptide pheromone transporter STE6 (9).The exact mechanism of P-gp action remains largely unsolved. A considerable body of evidence suggests that P-gp acts as a membrane-bound, ATP-dependent drug efflux pump that reduces intracellular drug accumulation in resistant cells. These include the observations that (i) P-gp expression in mdr transfectants causes both reduced accumulation and increased cellular efflux of MDR drugs, both of which are ATP dependent (1); (ii) P-gp binds photoactivatable ATP analogs (10) and shows ATPase activity (11), and mutations in either of its two predicted NB sites abrogate function (12); (iii) P-gp binds photoactivatable drug analogs (13), and mutations in its predicted transmembrane domains modulate substrate specificity and alter drug binding (14, 15); and (iv) P-gp shows structural and functional homology with a number of ATP-binding cassette transport proteins implicated in the transmembrane transport of structurally heterogeneous substrates, such as peptides, ions, sugars, and fatty acidsThe publication costs of this article were defrayed in p...

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Expression of the human multidrug transporter in insect cells by a recombinant baculovirus

Cited by 127 publications

References 42 publications

Comparisons of P‐glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta

Comparisons of P‐glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta

Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells

Functional expression of P-glycoprotein encoded by the mouse mdr3 gene in yeast cells.

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