Skip to main content
SpringerLink
Log in

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

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Similar content being viewed by others

References

  • N.G. Abdulaev T. Ngo R.W. Chen Z. Lu K.D. Ridge (2000) J. Biol. Chem. 275 IssueID50 39354–39363 Occurrence Handle10.1074/jbc.M005642200 Occurrence Handle10988291

    Article  PubMed  Google Scholar 

  • B. Antonny M. Chabre (1992) J. Biol. Chem. 267 IssueID10 6710–6718 Occurrence Handle1551879

    PubMed  Google Scholar 

  • F. Baneyx (1999) Curr. Opin. Biotech. 10 IssueID5 411–421 Occurrence Handle10.1016/S0958-1669(99)00003-8 Occurrence Handle10508629

    Article  PubMed  Google Scholar 

  • D.R. Benjamin D.W. Markby H.R. Bourne I.D. Kuntz (1995a) J. Mol. Biol. 254 IssueID4 681–691 Occurrence Handle10.1006/jmbi.1995.0647

    Article  Google Scholar 

  • D.R. Benjamin D.W. Markby H.R. Bourne I.D. Kuntz (1995b) Biochemistry 34 IssueID1 155–162 Occurrence Handle10.1021/bi00001a019

    Article  Google Scholar 

  • P.N. Bryan (2002) Chem. Rev. 102 IssueID12 4805–4815 Occurrence Handle10.1021/cr010190b Occurrence Handle12475207

    Article  PubMed  Google Scholar 

  • K. Cai Y. Itoh F.C. Khorana (2001) Proc. Natl. Acad. Sci. USA 98 IssueID9 4877–4882 Occurrence Handle10.1073/pnas.051632898 Occurrence Handle11320237

    Article  PubMed  Google Scholar 

  • D.E. Coleman A.M. Berghuis E. Lee M.E. Linder A.G. Gilman S.R. Sprang (1994) Science 265 IssueID5177 1405–1412 Occurrence Handle8073283

    PubMed  Google Scholar 

  • F. Delaglio S. Grzesiek G.W. Vuister G. Zhu J. Pfeifer A. Bax (1995) J. Biomol. NMR 6 IssueID3 277–293

    Google Scholar 

  • E. Faurobert A. Ottobruc P. Chardin M. Chabre (1993) EMBO J. 12 IssueID11 4191–4198 Occurrence Handle8223434

    PubMed  Google Scholar 

  • B.K.K. Fung J.B. Hurley L. Stryer (1981) Proc. Natl. Acad. Sci. USA 78 IssueID1 152–156 Occurrence Handle6264430

    PubMed  Google Scholar 

  • S. Grzesiek A. Bax (1993) J. Am. Chem. Soc. 115 IssueID26 12593–12594 Occurrence Handle10.1021/ja00079a052

    Article  Google Scholar 

  • P.M. Guy J.G. Koland R.A. Cerione (1990) Biochemistry 29 IssueID30 6954–6964 Occurrence Handle10.1021/bi00482a003 Occurrence Handle2223753

    Article  PubMed  Google Scholar 

  • H.E. Hamm (2001) Proc. Natl. Acad. Sci. USA 98 IssueID9 4819–4821 Occurrence Handle10.1073/pnas.011099798 Occurrence Handle11320227

    Article  PubMed  Google Scholar 

  • H.E. Hamm D. Deretic A. Arendt P.A. Hargrave B. Koenig K.P. Hofmann (1988) Science 241 IssueID4867 832–835 Occurrence Handle3136547

    PubMed  Google Scholar 

  • T. Higashijima M.P. Graziano H. Suga M. Kainosho A. Gilman (1991) J. Biol. Chem. 266 IssueID6 3396–3401 Occurrence Handle1899863

    PubMed  Google Scholar 

  • U.K. Laemmli (1970) Nature 277 IssueID5259 680–685

    Google Scholar 

  • D.G. Lambright J.P. Noel H.E. Hamm P.B. Sigler (1994) Nature 369 IssueID6482 621–628 Occurrence Handle10.1038/369621a0 Occurrence Handle8208289

    Article  PubMed  Google Scholar 

  • D.G. Lambright J. Sondek A. Bohm N.P. Skiba H.E. Hamm P.B. Sigler (1996) Nature 379 IssueID6563 311–319 Occurrence Handle10.1038/379311a0 Occurrence Handle8552184

    Article  PubMed  Google Scholar 

  • J. Li P.C. Edwards M. Burghammer C. Villa G.F. Schertler (2004) J. Mol. Biol. 343 IssueID5 1409–1438 Occurrence Handle10.1016/j.jmb.2004.08.090 Occurrence Handle15491621

    Article  PubMed  Google Scholar 

  • M.B. Mixon E. Lee D.E. Coleman A.M. Berghuis A.G. Gilman S.R. Sprang (1995) Science 270 IssueID5238 954–960 Occurrence Handle7481799

    PubMed  Google Scholar 

  • M. Natochin A.E. Granovsky K.G. Muradov N.O. Artemyev (1999) J. Biol. Chem. 274 IssueID12 7865–7869 Occurrence Handle10.1074/jbc.274.12.7865 Occurrence Handle10075679

    Article  PubMed  Google Scholar 

  • J.P. Noel H.E. Hamm P.B. Sigler (1993) Nature 366 IssueID6456 654–663 Occurrence Handle10.1038/366654a0 Occurrence Handle8259210

    Article  PubMed  Google Scholar 

  • T. Okada Y. Fujiyoshi M. Silow J. Navarro E.M. Landau Y. Shichida (2002) Proc. Natl. Acad. Sci. USA 99 IssueID9 5982–5987 Occurrence Handle10.1073/pnas.082666399 Occurrence Handle11972040

    Article  PubMed  Google Scholar 

  • T. Okada M. Sugihara A.N. Bondar M. Elstner P. Entel V. Buss (2004) J. Mol. Biol. 342 IssueID2 571–583 Occurrence Handle10.1016/j.jmb.2004.07.044 Occurrence Handle15327956

    Article  PubMed  Google Scholar 

  • J.O. Ortiz J. Bubis (2001) Arch. Biochem. Biophys. 387 IssueID2 233–242 Occurrence Handle10.1006/abbi.2000.2219 Occurrence Handle11370846

    Article  PubMed  Google Scholar 

  • K. Palczewski T. Kumasaka T. Hori C.A. Behnke H. Motoshima B.A. Fox I. Le Trong D.C. Teller T. Okada R.E. Stenkamp M. Yamamoto M. Miyano (2000) Science 289 IssueID5480 739–745 Occurrence Handle10.1126/science.289.5480.739 Occurrence Handle10926528

    Article  PubMed  Google Scholar 

  • K. Pervushin R. Riek G. Wider K. Wuthrich (1997) Proc. Natl. Acad. Sci. USA 94 IssueID23 12366–12371 Occurrence Handle10.1073/pnas.94.23.12366 Occurrence Handle9356455

    Article  PubMed  Google Scholar 

  • G.L. Peterson (1977) Anal. Biochem. 83 346–356 Occurrence Handle10.1016/0003-2697(77)90043-4 Occurrence Handle603028

    Article  PubMed  Google Scholar 

  • W.J. Phillips R.A. Cerione (1988) J. Biol. Chem. 263 IssueID30 15498–15505 Occurrence Handle3049609

    PubMed  Google Scholar 

  • J. Porath J. Carlsson I. Olsson G. Belfrage (1975) Nature 258 IssueID5536 598–599 Occurrence Handle1678

    PubMed  Google Scholar 

  • J. Reichert K.P. Hofmann (1984) FEBS Lett. 168 IssueID1 121–124 Occurrence Handle10.1016/0014-5793(84)80219-7 Occurrence Handle6705917

    Article  PubMed  Google Scholar 

  • K.D. Ridge N.G. Abdulaev M. Sousa K. Palczewski (2003) TIBS 28 IssueID9 479–487 Occurrence Handle13678959

    PubMed  Google Scholar 

  • B. Ruan K.E. Fisher P.A. Alexander V. Doroshko P.N. Bryan (2004) Biochemistry 43 IssueID46 14539–14546 Occurrence Handle10.1021/bi048177j Occurrence Handle15544324

    Article  PubMed  Google Scholar 

  • J. Schmitt H. Hess H.G. Stunnenberg (1993) Mol. Biol. Rep. 18 IssueID3 223–230 Occurrence Handle10.1007/BF01674434 Occurrence Handle8114690

    Article  PubMed  Google Scholar 

  • N.P. Skiba H. Bae H.E. Hamm (1996) J. Biol. Chem. 271 IssueID1 413–424 Occurrence Handle10.1074/jbc.271.1.413 Occurrence Handle8550597

    Article  PubMed  Google Scholar 

  • K.C. Slep M.A. Kercher W. He C.W. Cowan T.G. Wensel P.B. Sigler (2001) Nature 409 IssueID6823 1071–1077 Occurrence Handle10.1038/35059138 Occurrence Handle11234020

    Article  PubMed  Google Scholar 

  • J. Sondek D.G. Lambright J.P. Noel H.E. Hamm P.B. Sigler (1994) Nature 372 IssueID6503 276–279 Occurrence Handle10.1038/372276a0 Occurrence Handle7969474

    Article  PubMed  Google Scholar 

  • R.K. Sunahara J.J.G. Tesmer A.G. Gilman S.R. Sprang (1997) Science 278 IssueID5345 1943–1947 Occurrence Handle10.1126/science.278.5345.1943 Occurrence Handle9395396

    Article  PubMed  Google Scholar 

  • D.C. Teller T. Okada C.A. Behnke K. Palczewski R.E. Stenkamp (2001) Biochemistry 40 IssueID26 7761–7772 Occurrence Handle10.1021/bi0155091 Occurrence Handle11425302

    Article  PubMed  Google Scholar 

  • M.A. Wall D.E. Coleman E. Lee J.A. Iniguezlluhi B.A. Posner A.G. Gilman S.R. Sprang (1995) Cell 83 IssueID6 1047–1058 Occurrence Handle10.1016/0092-8674(95)90220-1 Occurrence Handle8521505

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, 20850, Rockville, MD, USA

    Najmoutin G. Abdulaev, Tony Ngo, Philip N. Bryan & John P. Marino

  2. National Institute of Standards and Technology, 20850, Rockville, MD, USA

    Najmoutin G. Abdulaev, Tony Ngo, Philip N. Bryan & John P. Marino

  3. Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, 77030, Houston, TX, USA

    Cheng Zhang, Andy Dinh & Kevin D. Ridge

  4. Department of Chemistry, Loyola College in Maryland, 21210, Baltimore, MD, USA

    Danielle M. Brabazon

Authors
  1. Najmoutin G. Abdulaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cheng Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Andy Dinh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tony Ngo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Philip N. Bryan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Danielle M. Brabazon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John P. Marino
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kevin D. Ridge
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John P. Marino.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Abdulaev, N., Zhang, C., Dinh, A. et al. Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein α-subunit. J Biomol NMR 32, 31–40 (2005). https://doi.org/10.1007/s10858-005-3340-5

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-005-3340-5

Key words:

Search

Navigation