Overexpression of homogeneous and active ABCG2 in insect cells.
The multidrug transporter ABCG2, a membrane protein with six transmembrane segments, can be overexpressed with the baculovirus/insect cell system. However, ABCG2 is produced as two species with different migration behavior via SDS-PAGE. Evidences suggest that this is due to the accumulation of an immature ABCG2 species, since: (i) the upper species, with higher apparent molecular weight, was favored by treatments reducing the rate of protein synthesis; (ii) the lower species was accumulated in presence of an endoplasmic reticulum stress inducer, and could be converted into the upper species during electrophoresis with 9 M urea; (iii) each species was differently solubilized by detergents: the upper species was partially solubilized by non-ionic and zwitterionic detergents, whereas the lower one required stronger surfactants; (iv) membrane ATPase activity from infected insect cells was essentially associated to the upper species. Altogether, these results suggest that although the insect cell/baculovirus system is not ideally adapted to overexpress human ABCG2, it is able to produce appreciable amounts of purified protein and the addition of agents reducing the rate of protein synthesis improves the homogeneity, making it a suitable heterologous expression system.
Pozza
Alexandre
A
Protéines de résistance aux agents chimiothérapeutiques, Institut de Biologie et Chimie des Protéines, UMR5086 CNRS-Université Lyon 1, Lyon, France.
Pérez-Victoria
José M
JM
Préz-Victoria
José M
JM
Di Pietro
Attilio
A
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D003513:Cycloheximide / Q000494:pharmacology; D019286:Endopeptidase K / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007313:Insecta / Q000166:cytology; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000187:drug effects / Q000302:isolation & purification*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D003513:Cycloheximide; D019286:Endopeptidase K