Abstract
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.
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References
Jarvis DL, Finn EE (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat Biotechnol 14:1288–92
van Meer G, Voelker DR, Feigenson GW (2008) Membrane lipids: where they are and how they behave. Nat Rev Mol Cell Biol 9:112–124
Kost TA, Condreay JP, Jarvis DL (2005) Baculovirus as versatile vectors for protein expression in insect and mammalian cells. Nat Biotech 23:567–575
Blissard GW, Rohrmann GF (1990) Baculovirus diversity and molecular biology. Annu Rev Entomol 35:127–155
Smith GE, Vlak JM, Summers MD (1982) In vitro translation of Autographa californica nuclear polyhedrosis virus early and late mRNAs. J Virol 44:199–208
Jarvis DL, Kawar ZS, Hollister JR (1998) Engineering N-glycosylation pathways in the baculovirus-insect cell system. Curr Opin Biotechnol 9:528–533
Marheineke K, Grunewald S, Christie W, Reilander H (1998) Lipid composition of Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) insect cells used for baculovirus infection. FEBS Lett 441:49–52
Gilbert RS, Nagano Y, Yokota T, Hwan SF, Fletcher T, Lydersen K (1996) Effect of lipids on insect cell growth and expression of recombinant proteins in serum-free medium. Cytotechnology 22:211–216
Dean M, Hamon Y, Chimini G (2001) The human ATP-binding cassette (ABC) transporter superfamily. J Lipid Res 42:1007–1017
Ross DD, Yang W, Abruzzo LV, Dalton WS, Schneider E, Lage H, Dietel M, Greenberger L, Cole SP, Doyle LA (1999) Atypical multidrug resistance: breast cancer resistance protein messenger RNA expression in mitoxantrone-selected cell lines. J Natl Cancer Inst 91:429–433
Litman T, Brangi M, Hudson E, Fetsch P, Abati A, Ross DD, Miyake K, Resau JH, Bates SE (2000) The multidrug-resistant phenotype associated with overexpression of the new ABC half-transporter, MXR (ABCG2). J Cell Sci 113(Pt 11):2011–2021
Ozvegy C, Varadi A, Sarkadi B (2002) Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation. J Biol Chem 277:47980–47990
McDevitt CA, Collins RF, Conway M, Modok S, Storm J, Kerr ID, Ford RC, Callaghan R (2006) Purification and 3D structural analysis of oligomeric human multidrug transporter ABCG2. Structure 14:1623–1632
Pozza A, Perez-Victoria JM, Sardo A, Ahmed-Belkacem A, Di Pietro A (2006) Direct interaction with purified breast cancer resistance protein ABCG2 indicates arginine-482 involvement in drug transport, not in binding. Cell Mol life Sci 63:1912–1922
Baker EK, Colley NJ, Zuker CS (1994) The cyclophilin homolog NinaA functions as a chaperone, forming a stable complex in vivo with its protein target rhodopsin. EMBO J 13:4886–4895
Lenhard T, Reilander H (1997) Engineering the folding pathway of insect cells: generation of a stably transformed insect cell line showing improved folding of a recombinant membrane protein. Biochem Biophys Res Commun 238:823–830
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, New York
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85
Pozza A, Perez-Victoria JM, Di Pietro A (2009) Overexpession of homogeneous and active ABCG2 in insect cells. Protein Expr Purif 63(2):75–83
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Trometer, C., Falson, P. (2010). Mammalian Membrane Protein Expression in Baculovirus-Infected Insect Cells. In: Mus-Veteau, I. (eds) Heterologous Expression of Membrane Proteins. Methods in Molecular Biology™, vol 601. Humana Press. https://doi.org/10.1007/978-1-60761-344-2_7
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DOI: https://doi.org/10.1007/978-1-60761-344-2_7
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