Advertisement

Journal of Biomolecular NMR

, Volume 32, Issue 1, pp 31–40 | Cite as

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein α-subunit

  • Najmoutin G. Abdulaev
  • Cheng Zhang
  • Andy Dinh
  • Tony Ngo
  • Philip N. Bryan
  • Danielle M. Brabazon
  • John P. MarinoEmail author
  • Kevin D. Ridge
Article

Abstract

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein α-subunit (Gα) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled Gα chimera (∼40 kDa polypeptide) has been tested. The results show that a prodomain fused Gα chimera can be expressed to levels approaching 6–8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of Gα isolated from natural sources. To assay for the functional integrity of the purified Gα chimera at NMR concentrations and probe for changes in the structure and dynamics of Gα that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of Gα obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with Gα activation.

Key words:

eukaryotic protein G-protein high-resolution stable-isotope labeling subtilisin transducin 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Abdulaev, N.G., Ngo, T., Chen, R.W., Lu, Z., Ridge, K.D. 2000J. Biol. Chem.2753935439363CrossRefPubMedGoogle Scholar
  2. Antonny, B., Chabre, M. 1992J. Biol. Chem.26767106718PubMedGoogle Scholar
  3. Baneyx, F. 1999Curr. Opin. Biotech.10411421CrossRefPubMedGoogle Scholar
  4. Benjamin, D.R., Markby, D.W., Bourne, H.R., Kuntz, I.D. 1995aJ. Mol. Biol.254681691CrossRefGoogle Scholar
  5. Benjamin, D.R., Markby, D.W., Bourne, H.R., Kuntz, I.D. 1995bBiochemistry34155162CrossRefGoogle Scholar
  6. Bryan, P.N. 2002Chem. Rev.10248054815CrossRefPubMedGoogle Scholar
  7. Cai, K., Itoh, Y., Khorana, F.C. 2001Proc. Natl. Acad. Sci. USA9848774882CrossRefPubMedGoogle Scholar
  8. Coleman, D.E., Berghuis, A.M., Lee, E., Linder, M.E., Gilman, A.G., Sprang, S.R. 1994Science26514051412PubMedGoogle Scholar
  9. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., Bax, A. 1995J. Biomol. NMR6277293Google Scholar
  10. Faurobert, E., Ottobruc, A., Chardin, P., Chabre, M. 1993EMBO J.1241914198PubMedGoogle Scholar
  11. Fung, B.K.K., Hurley, J.B., Stryer, L. 1981Proc. Natl. Acad. Sci. USA78152156PubMedGoogle Scholar
  12. Grzesiek, S., Bax, A. 1993J. Am. Chem. Soc.1151259312594CrossRefGoogle Scholar
  13. Guy, P.M., Koland, J.G., Cerione, R.A. 1990Biochemistry2969546964CrossRefPubMedGoogle Scholar
  14. Hamm, H.E. 2001Proc. Natl. Acad. Sci. USA9848194821CrossRefPubMedGoogle Scholar
  15. Hamm, H.E., Deretic, D., Arendt, A., Hargrave, P.A., Koenig, B., Hofmann, K.P. 1988Science241832835PubMedGoogle Scholar
  16. Higashijima, T., Graziano, M.P., Suga, H., Kainosho, M., Gilman, A. 1991J. Biol. Chem.26633963401PubMedGoogle Scholar
  17. Laemmli, U.K. 1970Nature277680685Google Scholar
  18. Lambright, D.G., Noel, J.P., Hamm, H.E., Sigler, P.B. 1994Nature369621628CrossRefPubMedGoogle Scholar
  19. Lambright, D.G., Sondek, J., Bohm, A., Skiba, N.P., Hamm, H.E., Sigler, P.B. 1996Nature379311319CrossRefPubMedGoogle Scholar
  20. Li, J., Edwards, P.C., Burghammer, M., Villa, C., Schertler, G.F. 2004J. Mol. Biol.34314091438CrossRefPubMedGoogle Scholar
  21. Mixon, M.B., Lee, E., Coleman, D.E., Berghuis, A.M., Gilman, A.G., Sprang, S.R. 1995Science270954960PubMedGoogle Scholar
  22. Natochin, M., Granovsky, A.E., Muradov, K.G., Artemyev, N.O. 1999J. Biol. Chem.27478657869CrossRefPubMedGoogle Scholar
  23. Noel, J.P., Hamm, H.E., Sigler, P.B. 1993Nature366654663CrossRefPubMedGoogle Scholar
  24. Okada, T., Fujiyoshi, Y., Silow, M., Navarro, J., Landau, E.M., Shichida, Y. 2002Proc. Natl. Acad. Sci. USA9959825987CrossRefPubMedGoogle Scholar
  25. Okada, T., Sugihara, M., Bondar, A.N., Elstner, M., Entel, P., Buss, V. 2004J. Mol. Biol.342571583CrossRefPubMedGoogle Scholar
  26. Ortiz, J.O., Bubis, J. 2001Arch. Biochem. Biophys.387233242CrossRefPubMedGoogle Scholar
  27. Palczewski, K., Kumasaka, T., Hori, T., Behnke, C.A., Motoshima, H., Fox, B.A., Le Trong, I., Teller, D.C., Okada, T., Stenkamp, R.E., Yamamoto, M., Miyano, M. 2000Science289739745CrossRefPubMedGoogle Scholar
  28. Pervushin, K., Riek, R., Wider, G., Wuthrich, K. 1997Proc. Natl. Acad. Sci. USA941236612371CrossRefPubMedGoogle Scholar
  29. Peterson, G.L. 1977Anal. Biochem.83346356CrossRefPubMedGoogle Scholar
  30. Phillips, W.J., Cerione, R.A. 1988J. Biol. Chem.2631549815505PubMedGoogle Scholar
  31. Porath, J., Carlsson, J., Olsson, I., Belfrage, G. 1975Nature258598599PubMedGoogle Scholar
  32. Reichert, J., Hofmann, K.P. 1984FEBS Lett.168121124CrossRefPubMedGoogle Scholar
  33. Ridge, K.D., Abdulaev, N.G., Sousa, M., Palczewski, K. 2003TIBS28479487PubMedGoogle Scholar
  34. Ruan, B., Fisher, K.E., Alexander, P.A., Doroshko, V., Bryan, P.N. 2004Biochemistry431453914546CrossRefPubMedGoogle Scholar
  35. Schmitt, J., Hess, H., Stunnenberg, H.G. 1993Mol. Biol. Rep.18223230CrossRefPubMedGoogle Scholar
  36. Skiba, N.P., Bae, H., Hamm, H.E. 1996J. Biol. Chem.271413424CrossRefPubMedGoogle Scholar
  37. Slep, K.C., Kercher, M.A., He, W., Cowan, C.W., Wensel, T.G., Sigler, P.B. 2001Nature40910711077CrossRefPubMedGoogle Scholar
  38. Sondek, J., Lambright, D.G., Noel, J.P., Hamm, H.E., Sigler, P.B. 1994Nature372276279CrossRefPubMedGoogle Scholar
  39. Sunahara, R.K., Tesmer, J.J.G., Gilman, A.G., Sprang, S.R. 1997Science27819431947CrossRefPubMedGoogle Scholar
  40. Teller, D.C., Okada, T., Behnke, C.A., Palczewski, K., Stenkamp, R.E. 2001Biochemistry4077617772CrossRefPubMedGoogle Scholar
  41. Wall, M.A., Coleman, D.E., Lee, E., Iniguezlluhi, J.A., Posner, B.A., Gilman, A.G., Sprang, S.R. 1995Cell8310471058CrossRefPubMedGoogle Scholar

Copyright information

© Springer 2005

Authors and Affiliations

  • Najmoutin G. Abdulaev
    • 1
    • 2
  • Cheng Zhang
    • 3
  • Andy Dinh
    • 3
  • Tony Ngo
    • 1
    • 2
  • Philip N. Bryan
    • 1
    • 2
  • Danielle M. Brabazon
    • 4
  • John P. Marino
    • 1
    • 2
    Email author
  • Kevin D. Ridge
    • 3
  1. 1.Center for Advanced Research in BiotechnologyUniversity of Maryland Biotechnology InstituteRockvilleUSA
  2. 2.National Institute of Standards and TechnologyRockvilleUSA
  3. 3.Center for Membrane Biology, Department of Biochemistry and Molecular BiologyUniversity of Texas Health Science CenterHoustonUSA
  4. 4.Department of ChemistryLoyola College in MarylandBaltimoreUSA

Personalised recommendations