Abundant expression and purification of biologically active mitochondrial citrate carrier in baculovirus-infected insect cells

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.

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References

  1. Bisaccia F, De Palma A, Palmieri F (1989) Biochim Biophys Acta 977:171–176

    Article  CAS  Google Scholar 

  2. Capobianco L, Bisaccia F, Mazzeo M, Palmieri F (1996) Biochemistry 35:8974–8980

    Article  CAS  Google Scholar 

  3. Capobianco L, Ferramosca A, Zara V (2002) J Protein Chem 21:515–521

    Article  CAS  Google Scholar 

  4. Capobianco L, Impagnatiello T, Ferramosca A, Zara V (2004) J Biochem Mol Biol 37:337–353

    Google Scholar 

  5. Carrisi C, Madeo M, Morciano P, Dolce V, Cenci G, Cappello AR, Mazzeo G, Iacopetta D, Capobianco L (2008) J Biochem 144:389–392

    Article  CAS  Google Scholar 

  6. Conover TE (1987) Trends Biochem Sci 12:88–89

    Article  CAS  Google Scholar 

  7. Dolby V, Collén A, Lundqvist A, Cronet P (2004) Protein Expr Purif 37:455–461

    Article  CAS  Google Scholar 

  8. 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

    Article  CAS  Google Scholar 

  9. Fiermonte G, Walker JE, Palmieri F (1993) Biochem J 294:293–299

    CAS  Google Scholar 

  10. Fiermonte G, Dolce V, Palmieri F (1998) 273:22782-22787

  11. Goffart S, Martinsson P, Malka F, Rojo M, Spelbrink JN (2007) Methods Mol Biol 372:17–32

    Article  CAS  Google Scholar 

  12. Grisshammer R, Tate CG (1995) Q Rev Biophys 28:315–422

    Article  CAS  Google Scholar 

  13. Heimpel S, Basset G, Odoy S, Klingenberg M (2001) J Biol Chem 276:11499–11506

    Article  CAS  Google Scholar 

  14. Hung YH, Layton MJ, Voskoboinik I, Mercer JF, Camakaris J (2007) Biochem J 401:569–579

    Article  CAS  Google Scholar 

  15. Knecht W, Altekruse D, Rotgeri A, Gonski S, Löffler M (1997) Protein Expr Purif 10:89–99

    Article  CAS  Google Scholar 

  16. Kost TA, Condreay JP, Jarvis DL (2005) Nat Biotechnol 23:567–575

    Article  CAS  Google Scholar 

  17. Kramer R, Klingenberg M (1979) Biochemistry 18:4209–4215

    Article  CAS  Google Scholar 

  18. Loguercio Polosa P, Deceglie S, Falkenberg M, Roberti M, Di Ponzio B, Gadaleta MN, Cantatore P (2007) Nucleic Acids Res 35:2413–2427

    Article  Google Scholar 

  19. Meijer AJ, Van Dam K (1974) Biochem Biophys Acta 346:213–244

    CAS  Google Scholar 

  20. Miroux B, Walker JE (1996) J Mol Biol 260:289–298

    Article  CAS  Google Scholar 

  21. Mitic LL, Unger VM, Anderson JM (2003) Protein Sci 12:218–227

    Article  CAS  Google Scholar 

  22. O’Reilly DR, Miller LK, Luckow VA (1992) Baculovirus Expression Vectors: A Laboratory Manual. Oxford University Press, New York

    Google Scholar 

  23. Palmieri F (2004) Pflugers Arch–Eur J Physiol 447:689–709

    Article  CAS  Google Scholar 

  24. Palmieri F (2008) Biochim Biophys Acta 1777:564–578

    Article  CAS  Google Scholar 

  25. Palmieri F, Stipani I, Quagliariello E, Klingenberg M (1972) Eur J Biochem 26:587–594

    Article  CAS  Google Scholar 

  26. Palmieri F, Indiveri C, Bisaccia F, Krämer R (1993) J Bioenerg Biomembr 25:525–535

    Article  CAS  Google Scholar 

  27. Palmieri F, Indiveri C, Bisaccia F, Iacobazzi V (1995) Methods Enzymol 260:349–369

    Article  CAS  Google Scholar 

  28. Palmieri L, Agrimi G, Runswick MJ, Fearnley IM, Palmieri F, Walker JE (2001) J Biol Chem 276:1916–1922

    CAS  Google Scholar 

  29. 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

    Article  CAS  Google Scholar 

  30. Palmieri L, Arrigoni R, Blanco E, Carrari F, Zanor MI, Studart-Guimareas C, Fernie AR, Palmieri F (2006b) Plant Physiol 142:855–865

    Article  CAS  Google Scholar 

  31. Palmieri L, Picault N, Arrigoni R, Besin E, Palmieri F, Hodges M (2008) Biochem J 410:621–629

    Article  CAS  Google Scholar 

  32. Picault N, Hodges M, Palmieri L, Palmieri F (2004) Trends in Plant Sci 9:138–146

    Article  CAS  Google Scholar 

  33. Ratnala VR, Swarts HG, VanOostrum J, Leurs R, DeGroot HJ, Bakker RA, DeGrip WJ (2004) Eur J Biochem 271:2636–2646

    Article  CAS  Google Scholar 

  34. Rosenbusch JP (2001) J Struct Biol 136:144–157

    Article  CAS  Google Scholar 

  35. Sharp PM, Cowe E, Higgins DG, Shields DC, Wolfe KH, Wright F (1988) Nucleic Acids Res 16:8207–8211

    Article  CAS  Google Scholar 

  36. Shi X, Jarvis DL (2007) Curr Drug Targets 8:1116–1125

    Article  CAS  Google Scholar 

  37. Smith GE, Summers MD, Fraser MJ (1983) Mol Cell Biol 3:2156–2165

    CAS  Google Scholar 

  38. Smith GE, Summers MD, Fraser MJ (1992) Biotechnology 24:434–443

    CAS  Google Scholar 

  39. Wagner S, Bader ML, Drew D, de Gier JW (2006) Trends Biotechnol 24:364–371

    Article  CAS  Google Scholar 

  40. Winkler E, Heidkaemper D, Klingenberg M, Liu Q, Caskey T (2001) Biochem Biophys Res Commun 282:334–340

    Article  CAS  Google Scholar 

  41. Zara V, Iacobazzi V, Siculella L, Gnoni GV, Palmieri F (1996) Biochem Biophys Res Commun 223:508–513

    Article  CAS  Google Scholar 

  42. Zara V, Palmieri L, Franco MR, Perrone M, Gnoni GV, Palmieri F (1998) J Bioenerg Biomembr 30:555–563

    Article  CAS  Google Scholar 

  43. Zara V, Palmieri L, Giudetti A, Ferramosca A, Capobianco L, Gnoni GV (2000) Biochem Biophys Res Commun 276:893–898

    Article  CAS  Google Scholar 

  44. Zara V, Dolce V, Capobianco L, Ferramosca A, Papatheodorou P, Rassow J, Palmieri F (2007) J Mol Biol 365:958–967

    Article  CAS  Google Scholar 

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Correspondence to Loredana Capobianco or Vincenza Dolce.

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Marianna Madeo and Chiara Carrisi contributed equally to this work.

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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

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Keywords

  • Mitochondria
  • Citrate carrier
  • Membrane protein
  • Detergents
  • V5/His-tag
  • Baculovirus expression