ABCMdb

PMID: 20665261

Pozza A, Perez-Victoria JM, Di Pietro A
Insect cell versus bacterial overexpressed membrane proteins: an example, the human ABCG2 transporter.
Methods Mol Biol. 2010;654:47-75., [PubMed]

Sentences

No. Mutations Sentence Comment

49 ABCG2 p.Arg482Thr The pcDNA3 plasmid containing R482T-ABCG2 cDNA was kindly provided by Dr. D. Login to comment 52 ABCG2 p.Arg482Thr Site-directed mutagenesis: The R482T-ABCG2 cDNA was mutated to obtain R482-ABCG2 cDNA by site-directed mutagenesis using a "Quick-Change Site-directed mutagenesis" kit (Stratagene, La Jolla, CA). Login to comment 147 ABCG2 p.Arg482Thr Circular dichroism detergent buffer: 10 mM NaPO4 , pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (either SDS for ABCG2 R482T from bacteria or dodecylmaltoside for the protein from insect cells). Login to comment 151 ABCG2 p.Arg482Thr E. coli strains were transformed with pET21b(+)/ABCG2 R482T using a thermal-shock procedure. Login to comment 191 ABCG2 p.Arg482Thr 10. Finally, cured PV6 mutants were transformed with pET21b(+)/ABCG2 R482T, and protein expression and growth kinetics were monitored. Login to comment 193 ABCG2 p.Arg482Thr 10. Finally, cured PV6 mutants were transformed with pET21b(+)/ABCG2 R482T, and protein expression and growth kinetics were monitored. Login to comment 208 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr (■), BL21(DE3)/pET21a(+) ABCG2 R482T; (§), BL21(DE3)/pET21a(+) ABCG2 R482T induced; (▲), PV6/pET21a(+) ABCG2 R482T; ( ), PV6/pET21a(+) ABCG2 R482T induced. Login to comment 211 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr (■), BL21(DE3)/pET21a(+) ABCG2 R482T; (§), BL21(DE3)/pET21a(+) ABCG2 R482T induced; (▲), PV6/pET21a(+) ABCG2 R482T; ( ), PV6/pET21a(+) ABCG2 R482T induced. Login to comment 361 ABCG2 p.Lys86Met The basal ATPase activity was determined on inverted membrane vesicles from control insect cells expressing b-galactosidase or inactive K86M ABCG2. Login to comment 364 ABCG2 p.Lys86Met The basal ATPase activity was determined on inverted membrane vesicles from control insect cells expressing b-galactosidase or inactive K86M ABCG2. Login to comment 371 ABCG2 p.Arg482Thr (b) Effects of substrates and inhibitors on the ATPase activity of ABCG2 within the inverted membrane vesicles from insect cells.TheATPase activity of R482 (black columns) or R482T (gray columns) ABCG2 overexpressed in Sf9 was measured with 2.5 µg of protein, 5 mM ATP, and 20 mM MgCl2 at 37°C for 40 min. Login to comment 374 ABCG2 p.Arg482Thr (b) Effects of substrates and inhibitors on the ATPase activity of ABCG2 within the inverted membrane vesicles from insect cells.TheATPase activity of R482 (black columns) or R482T (gray columns) ABCG2 overexpressed in Sf9 was measured with 2.5 µg of protein, 5 mM ATP, and 20 mM MgCl2 at 37°C for 40 min. Login to comment 392 ABCG2 p.Arg482Thr Inverted membrane vesicles from High-Five cells infected by a baculovirus vector encoding the R482 or R482T transporter were treated with cell solubilization buffer at a final protein concentration of 2 mg/ml, for 30 min with gente shaking at 4°C. Login to comment 395 ABCG2 p.Arg482Thr Inverted membrane vesicles from High-Five cells infected by a baculovirus vector encoding the R482 or R482T transporter were treated with cell solubilization buffer at a final protein concentration of 2 mg/ml, for 30 min with gente shaking at 4°C. Login to comment 421 ABCG2 p.Arg482Thr Lane 1: prestained molecular weight markers; lane 2 : inverted membrane vesicles of High-Five cells infected with the baculovirus vector encoding either R482 or R482T ABCG2; lane 3 : supernatant after solubilization; lane 4: supernatant after centrifugation (15,000 × g, 30 min); lane 5 : detergent-insoluble pellet after centrifugation; lane 6 : supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8 : washing; lane 9 : Ni-NTA agarose gel after washing; lane 10 : elution; lane 11: Ni-NTA agarose gel after elution; lane 12 : after imidazole removal by gel filtration. Login to comment 424 ABCG2 p.Arg482Thr Lane 1: prestained molecular weight markers; lane 2 : inverted membrane vesicles of High-Five cells infected with the baculovirus vector encoding either R482 or R482T ABCG2; lane 3 : supernatant after solubilization; lane 4: supernatant after centrifugation (15,000 × g, 30 min); lane 5 : detergent-insoluble pellet after centrifugation; lane 6 : supernatant after binding for 2 h; lane 7: Ni-NTA agarose gel after binding for 2 h; lane 8 : washing; lane 9 : Ni-NTA agarose gel after washing; lane 10 : elution; lane 11: Ni-NTA agarose gel after elution; lane 12 : after imidazole removal by gel filtration. Login to comment 440 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Measurements were performed under the following conditions: 10 mM NaPi, pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (SDS for 0.5 µM ABCG2 R482T from bacteria or dodecylmaltoside for 0.24 µM ABCG2 R482T from insect cells), and spectra was scanned from 185-190 to 250 nm with 0.2-nm step and 1-s data acquisition time at 25°C. Login to comment 441 ABCG2 p.Arg482Thr (c) Binding of 6-prenylchrysin on purified ABCG2 R482T from bacteria or insect cells. Login to comment 442 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr The measurements were performed under the following conditions: 0.5 µM ABCG2 R482T, 0.05% SDS, 0.1 M KPi, 15% glycerol, 0.1 M NaCl, 1 mM DTT at 25°C for ABCG2 from bacteria (■), and 0.6 µM ABCG2 R482T, 50 mM HEPES/NaOH, pH 8, 18 mM CHAPS, 0.5 M NaCl, 20% glycerol ABCG2 for insect cell-expressed transporter (§). Login to comment 443 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr Measurements were performed under the following conditions: 10 mM NaPi, pH 8, 2% glycerol, 1 mM DTT, 0.05% detergent (SDS for 0.5 µM ABCG2 R482T from bacteria or dodecylmaltoside for 0.24 µM ABCG2 R482T from insect cells), and spectra was scanned from 185-190 to 250 nm with 0.2-nm step and 1-s data acquisition time at 25°C. Login to comment 444 ABCG2 p.Arg482Thr (c) Binding of 6-prenylchrysin on purified ABCG2 R482T from bacteria or insect cells. Login to comment 445 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr The measurements were performed under the following conditions: 0.5 µM ABCG2 R482T, 0.05% SDS, 0.1 M KPi, 15% glycerol, 0.1 M NaCl, 1 mM DTT at 25°C for ABCG2 from bacteria (■), and 0.6 µM ABCG2 R482T, 50 mM HEPES/NaOH, pH 8, 18 mM CHAPS, 0.5 M NaCl, 20% glycerol ABCG2 for insect cell-expressed transporter (§). Login to comment 446 ABCG2 p.Arg482Thr (d) Inhibition by vanadate of the ATPase activity of purified ABCG2 from insect cells.The inhibition of ATPase activity of R482 (squares) and R482T (triangles) transporter was measured in the presence of 5 mM MgATP, 0.5 µg protein, 50 µg azolectin, and a range of orthovanade concentrations from 0.001 to 10 mM at 37°C for 40 min. Login to comment 448 ABCG2 p.Arg482Thr The ATPase activity of either R482 (squares) or R482T (triangles) purified ABCG2 was measured with 0.5 µg protein in the presence of 50 µg azolectin at 37°C for 40 min. Login to comment 449 ABCG2 p.Arg482Thr (d) Inhibition by vanadate of the ATPase activity of purified ABCG2 from insect cells.The inhibition of ATPase activity of R482 (squares) and R482T (triangles) transporter was measured in the presence of 5 mM MgATP, 0.5 µg protein, 50 µg azolectin, and a range of orthovanade concentrations from 0.001 to 10 mM at 37°C for 40 min. Login to comment 450 ABCG2 p.Arg482Thr Measurements were performed under the following conditions:0.045 µg/µlABCG2 (either R482 or R482T),50 mM HEPES/NaOH,pH 8,18 mM CHAPS,0.5 M NaCl, 20% glycerol. Login to comment 451 ABCG2 p.Arg482Thr The ATPase activity of either R482 (squares) or R482T (triangles) purified ABCG2 was measured with 0.5 µg protein in the presence of 50 µg azolectin at 37°C for 40 min. Login to comment 453 ABCG2 p.Arg482Thr Measurements were performed under the following conditions:0.045 µg/µlABCG2 (either R482 or R482T),50 mM HEPES/NaOH,pH 8,18 mM CHAPS,0.5 M NaCl, 20% glycerol. Login to comment 478 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr The apparent Kd values were not modified by the R482T point mutation (Fig. 4f), suggesting that the R482T point mutation does not act on binding but on subsequent step to transport. Login to comment 481 ABCG2 p.Arg482Thr ABCG2 p.Arg482Thr The apparent Kd values were not modified by the R482T point mutation (Fig. 4f), suggesting that the R482T point mutation does not act on binding but on subsequent step to transport. Login to comment 491 ABCG2 p.Arg482Thr The ATPase activity of purified ABCG2 was modified by the R482T point mutation, which both increased Vmax and decreased Km (ATP/Mg2+ ) (Fig. 4e). Login to comment 494 ABCG2 p.Arg482Thr The ATPase activity of purified ABCG2 was modified by the R482T point mutation, which both increased Vmax and decreased Km (ATP/Mg2+ ) (Fig. 4e). Login to comment