Mammalian membrane protein expression in baculovirus-infected insect cells.
A system of expression of mammalian membrane proteins in baculovirus-infected insect cells is described, allowing analytical or preparative production in the milligram range of such type of proteins. This is illustrated by the setup of the expression system of the human breast cancer resistance protein (BCRP), which is a homodimeric multidrug ABC (adenosine triphosphate-binding cassette) transporter. The system used is Bac to Bac, which allows generation of a viral genome that includes the protein of interest by using a shuttle plasmid and a bacterial host carrying the bacmid. In the present case, the use of a molecular chaperon, ninaA, is illustrated, co-expressed with BCRP by using a plasmid allowing the expression of two proteins, pFastBac Dual. The method is detailed to allow the expression of such proteins and membrane protein in general.
Trometer
Céline
C
Institut de Biologie et Chimie des Protéies, Unité Mixte du Centre National de la Recherche Scientifique et de l'Université Lyon, France.
Falson
Pierre
P
D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004330:Drosophila / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006801:Humans; D007313:Insecta / Q000166:cytology / Q000235:genetics / Q000821:virology*; D008565:Membrane Proteins / Q000235:genetics; D018832:Molecular Chaperones / Q000235:genetics; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D014162:Transfection
C116614:ABCG2 protein, human; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D029721:Drosophila Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; C068480:ninaA protein, Drosophila