Abstract
Heterologous expression of recombinant proteins is an essential technology for protein characterization. A major obstacle to investigating the biochemical properties of membrane proteins is the difficulty in obtaining sufficient amounts of functional protein. Here we report the successful expression of the tricarboxylate (or citrate) carrier (CIC) of eel (Anguilla anguilla) from Spodoptera frugiperda (Sf9) cells using the baculovirus expression system. The recombinant CIC was purified by affinity chromatography on Ni2+-NTA agarose; the yield of the purified active protein was 0.4–0.5 mg/l of culture. The transport characteristics of the recombinant CIC and the effects of inhibitors on transport are similar to those determined for eel liver mitochondrial CIC. Because the CIC is one member of an extensive family of mitochondrial transport proteins, it is likely that the procedure used in this study to express and purify this carrier can be successfully applied to other mitochondrial transport proteins, thus providing sufficient protein for functional characterization.
Similar content being viewed by others
References
Bisaccia F, De Palma A, Palmieri F (1989) Biochim Biophys Acta 977:171–176
Capobianco L, Bisaccia F, Mazzeo M, Palmieri F (1996) Biochemistry 35:8974–8980
Capobianco L, Ferramosca A, Zara V (2002) J Protein Chem 21:515–521
Capobianco L, Impagnatiello T, Ferramosca A, Zara V (2004) J Biochem Mol Biol 37:337–353
Carrisi C, Madeo M, Morciano P, Dolce V, Cenci G, Cappello AR, Mazzeo G, Iacopetta D, Capobianco L (2008) J Biochem 144:389–392
Conover TE (1987) Trends Biochem Sci 12:88–89
Dolby V, Collén A, Lundqvist A, Cronet P (2004) Protein Expr Purif 37:455–461
Eifler N, Duckely M, Sumanovski LT, Egan TM, Oksche A, Konopka JB, Lüthi A, Engel A, Werten PJ (2007) J Struct Biol 159:179–193
Fiermonte G, Walker JE, Palmieri F (1993) Biochem J 294:293–299
Fiermonte G, Dolce V, Palmieri F (1998) 273:22782-22787
Goffart S, Martinsson P, Malka F, Rojo M, Spelbrink JN (2007) Methods Mol Biol 372:17–32
Grisshammer R, Tate CG (1995) Q Rev Biophys 28:315–422
Heimpel S, Basset G, Odoy S, Klingenberg M (2001) J Biol Chem 276:11499–11506
Hung YH, Layton MJ, Voskoboinik I, Mercer JF, Camakaris J (2007) Biochem J 401:569–579
Knecht W, Altekruse D, Rotgeri A, Gonski S, Löffler M (1997) Protein Expr Purif 10:89–99
Kost TA, Condreay JP, Jarvis DL (2005) Nat Biotechnol 23:567–575
Kramer R, Klingenberg M (1979) Biochemistry 18:4209–4215
Loguercio Polosa P, Deceglie S, Falkenberg M, Roberti M, Di Ponzio B, Gadaleta MN, Cantatore P (2007) Nucleic Acids Res 35:2413–2427
Meijer AJ, Van Dam K (1974) Biochem Biophys Acta 346:213–244
Miroux B, Walker JE (1996) J Mol Biol 260:289–298
Mitic LL, Unger VM, Anderson JM (2003) Protein Sci 12:218–227
O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York
Palmieri F (2004) Pflugers Arch–Eur J Physiol 447:689–709
Palmieri F (2008) Biochim Biophys Acta 1777:564–578
Palmieri F, Stipani I, Quagliariello E, Klingenberg M (1972) Eur J Biochem 26:587–594
Palmieri F, Indiveri C, Bisaccia F, Krämer R (1993) J Bioenerg Biomembr 25:525–535
Palmieri F, Indiveri C, Bisaccia F, Iacobazzi V (1995) Methods Enzymol 260:349–369
Palmieri L, Agrimi G, Runswick MJ, Fearnley IM, Palmieri F, Walker JE (2001) J Biol Chem 276:1916–1922
Palmieri F, Agrimi G, Blanco E, Castegna A, Di Noia MA, Iacobazzi V, Lasorsa FM, Marobbio CMT, Palmieri L, Scarcia P, Todisco S, Vozza A, Walker J (2006a) Biochim Biophys Acta 1757:1249–1262
Palmieri L, Arrigoni R, Blanco E, Carrari F, Zanor MI, Studart-Guimareas C, Fernie AR, Palmieri F (2006b) Plant Physiol 142:855–865
Palmieri L, Picault N, Arrigoni R, Besin E, Palmieri F, Hodges M (2008) Biochem J 410:621–629
Picault N, Hodges M, Palmieri L, Palmieri F (2004) Trends in Plant Sci 9:138–146
Ratnala VR, Swarts HG, VanOostrum J, Leurs R, DeGroot HJ, Bakker RA, DeGrip WJ (2004) Eur J Biochem 271:2636–2646
Rosenbusch JP (2001) J Struct Biol 136:144–157
Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, Wright F (1988) Nucleic Acids Res 16:8207–8211
Shi X, Jarvis DL (2007) Curr Drug Targets 8:1116–1125
Smith GE, Summers MD, Fraser MJ (1983) Mol Cell Biol 3:2156–2165
Smith GE, Summers MD, Fraser MJ (1992) Biotechnology 24:434–443
Wagner S, Bader ML, Drew D, de Gier JW (2006) Trends Biotechnol 24:364–371
Winkler E, Heidkaemper D, Klingenberg M, Liu Q, Caskey T (2001) Biochem Biophys Res Commun 282:334–340
Zara V, Iacobazzi V, Siculella L, Gnoni GV, Palmieri F (1996) Biochem Biophys Res Commun 223:508–513
Zara V, Palmieri L, Franco MR, Perrone M, Gnoni GV, Palmieri F (1998) J Bioenerg Biomembr 30:555–563
Zara V, Palmieri L, Giudetti A, Ferramosca A, Capobianco L, Gnoni GV (2000) Biochem Biophys Res Commun 276:893–898
Zara V, Dolce V, Capobianco L, Ferramosca A, Papatheodorou P, Rassow J, Palmieri F (2007) J Mol Biol 365:958–967
Author information
Authors and Affiliations
Corresponding authors
Additional information
Marianna Madeo and Chiara Carrisi contributed equally to this work.
Rights and permissions
About this article
Cite this article
Madeo, M., Carrisi, C., Iacopetta, D. et al. Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells. J Bioenerg Biomembr 41, 289–297 (2009). https://doi.org/10.1007/s10863-009-9226-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10863-009-9226-6