Strategies for folding of affinity tagged proteins using GroEL and osmolytes

  • Hiroo Katayama
  • Mitchell McGill
  • Andrew Kearns
  • Marek Brzozowski
  • Nicholas Degner
  • Bliss Harnett
  • Boris Kornilayev
  • Dubravka Matković-Čalogović
  • Todd Holyoak
  • James P. Calvet
  • Edward P. Gogol
  • John Seed
  • Mark T. FisherEmail author


Obtaining a proper fold of affinity tagged chimera proteins can be difficult. Frequently, the protein of interest aggregates after the chimeric affinity tag is cleaved off, even when the entire chimeric construct is initially soluble. If the attached protein is incorrectly folded, chaperone proteins such as GroEL bind to the misfolded construct and complicate both folding and affinity purification. Since chaperonin/osmolyte mixtures facilitate correct folding from the chaperonin, we explored the possibility that we could use this intrinsic binding reaction to advantage to refold two difficult-to-fold chimeric constructs. In one instance, we were able to recover activity from a properly folded construct after the construct was released from the chaperonin in the presence of osmolytes. As an added advantage, we have also found that this method involving chaperonins can enable researchers to decide (1) if further stabilization of the folded product is required and (2) if the protein construct in question will ever be competent to fold with osmolytes.


Chaperonin Osmolytes Affinity tags Protein folding Protein stability 





Nickel-nitrilotriacetic acid


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Small ubiquitin-like modifier








Phosphoenolpyruvate carboxykinase





This research was supported in part by NSF MCB-0445936 (MTF), NIH R41 GM080074 (MTF), NIH P50 DK057301 (JPC), and a grant from the Kansas Technology Enterprise Corporation KTEC (MTF). MB and AK are undergraduates supported by the NSF grant. AK was specifically supported by an NSF REU supplement NSF MCB-0735909 (MTF). A portion of this research was presented at the “Protein Structure Inititative “Bottlenecks” Workshop. The authors gratefully acknowledge Stephen C. Parnell for generating the pSUMO-HT193 plasmid and for helpful discussions with D. M.-C. D. M.-C. was supported by a Fulbright Scholarship.


  1. 1.
    Thain A, Gaston K, Jenkins O, Clarke AR (1996) Trends Genet 12:209–210. doi: 10.1016/S0168-9525(96)90022-0 PubMedCrossRefGoogle Scholar
  2. 2.
    Horwich AL, Fenton WA (1997) Protein Sci 6:743–760PubMedGoogle Scholar
  3. 3.
    Fisher MT, Yuan X (1994) J Biol Chem 269:29598–29601PubMedGoogle Scholar
  4. 4.
    Weissman JS, Kashi Y, Fenton WA, Horwich AL (1994) Cell 78:693–702. doi: 10.1016/0092-8674(94)90533-9 PubMedCrossRefGoogle Scholar
  5. 5.
    Weissman JS, Rye HS, Fenton WA, Beechem JM, Horwich AL (1996) Cell 84:481–490. doi: 10.1016/S0092-8674(00)81293-3 PubMedCrossRefGoogle Scholar
  6. 6.
    Rye HS, Roseman AM, Chen S, Furtak K, Fenton WA, Saibil HR, Horwich AL (1999) Cell 97:325–328. doi: 10.1016/S0092-8674(00)80742-4 PubMedCrossRefGoogle Scholar
  7. 7.
    Mayhew M, da Silva ACR, Martin J, Erdjument-Bromage H, Tempst P, Hartl F-U (1996) Nature 379:420–426. doi: 10.1038/379420a0 PubMedCrossRefGoogle Scholar
  8. 8.
    Dubaquie Y, Looser R, Funfschilling U, Jeno P, Rospert S (1998) EMBO J 17:5868–5876. doi: 10.1093/emboj/17.20.5868 PubMedCrossRefGoogle Scholar
  9. 9.
    Chaudhuri TK, Farr GW, Fenton WA, Rospert S, Horwich AL (2001) Cell 107:235–246. doi: 10.1016/S0092-8674(01)00523-2 PubMedCrossRefGoogle Scholar
  10. 10.
    Sigler PB, Xu Z, Rye HS, Burston SG, Fenton WA, Horwich AL (1998) Annu Rev Biochem 67:581–608. doi: 10.1146/annurev.biochem.67.1.581 PubMedCrossRefGoogle Scholar
  11. 11.
    Voziyan PA, Jadhav L, Fisher MT (2000) J Pharm Sci 89:1036–1045. doi:10.1002/1520-6017(200008)89:8<1036::AID-JPS8>3.0.CO;2-5PubMedCrossRefGoogle Scholar
  12. 12.
    Tieman BC, Johnston M, Fisher MT (2001) J Biol Chem 276:44541–44550. doi: 10.1074/jbc.M106693200 PubMedCrossRefGoogle Scholar
  13. 13.
    Voziyan PA, Johnston M, Chao A, Bomhoff G, Fisher MT (2005) J Struct Funct Genomics 6:183–188. doi: 10.1007/s10969-005-2646-6 PubMedCrossRefGoogle Scholar
  14. 14.
    Katayama H, Janowiak BE, Brzozowski M, Jurcyk J, Falke S, Gogol EP, Collier RJ, Fisher MT (2008) Nat Struct Mol Biol 15:754–760. doi: 10.1038/nsmb.1442 PubMedCrossRefGoogle Scholar
  15. 15.
    Voziyan PA, Fisher MT (2000) Protein Sci 9:2405–2415PubMedCrossRefGoogle Scholar
  16. 16.
    Hebda CA, Nowak T (1982) J Biol Chem 257:5503–5514PubMedGoogle Scholar
  17. 17.
    Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A (1996) J Struct Biol 116:190–199. doi: 10.1006/jsbi.1996.0030 PubMedCrossRefGoogle Scholar
  18. 18.
    Fisher MT (1992) Biochemistry 31:3955–3963. doi: 10.1021/bi00131a010 PubMedCrossRefGoogle Scholar
  19. 19.
    Fisher MT (1994) J Biol Chem 269:13629–13636PubMedGoogle Scholar
  20. 20.
    Falke SF, Fisher MT, Gogol EG (2001) J Mol Biol 308:569–577. doi: 10.1006/jmbi.2001.4613 PubMedCrossRefGoogle Scholar
  21. 21.
    Falke SF, Fisher MT, Gogol EG (2001) J Struct Biol 133:203–213. doi: 10.1006/jsbi.2001.4354 PubMedCrossRefGoogle Scholar
  22. 22.
    Viitanen PV, Donaldson GK, Lorimer GH, Lubben TH, Gatenby AA (1991) Biochemistry 30:9716–9723. doi: 10.1021/bi00104a021 PubMedCrossRefGoogle Scholar
  23. 23.
    Smith KE, Voziyan PA, Fisher MT (1998) J Biol Chem 273:28677–28681. doi: 10.1074/jbc.273.44.28677 PubMedCrossRefGoogle Scholar
  24. 24.
    Walter S, Lorimer GH, Schmid FX (1996) Proc Natl Acad Sci USA 93:9425–9430. doi: 10.1073/pnas.93.18.9425 PubMedCrossRefGoogle Scholar
  25. 25.
    Holyoak T, Sullivan SM, Nowak T (2006) Biochemistry 45:8254–8263. doi: 10.1021/bi060269g PubMedCrossRefGoogle Scholar
  26. 26.
    Sot B, Bañuelos S, Valpuesta JM, Muga A (2003) J Biol Chem 278:32083–32090. doi: 10.1074/jbc.M303958200 PubMedCrossRefGoogle Scholar
  27. 27.
    Gallagher AR, Cedzich A, Gretz N, Somlo S, Witzgall R (2000) Proc Natl Acad Sci USA 97:4017–4022. doi: 10.1073/pnas.97.8.4017 PubMedCrossRefGoogle Scholar
  28. 28.
    Qian F, Germino J, Cai Y, Zhang X, Somlo S, Germino GG (1997) Nat Genet 16:179–183PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Hiroo Katayama
    • 1
  • Mitchell McGill
    • 1
  • Andrew Kearns
    • 1
  • Marek Brzozowski
    • 1
  • Nicholas Degner
    • 1
  • Bliss Harnett
    • 1
  • Boris Kornilayev
    • 1
  • Dubravka Matković-Čalogović
    • 1
    • 2
  • Todd Holyoak
    • 1
  • James P. Calvet
    • 1
  • Edward P. Gogol
    • 3
  • John Seed
    • 4
  • Mark T. Fisher
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Kansas Medical CenterKansas CityUSA
  2. 2.Department of Chemistry, Faculty of ScienceUniversity of ZagrebZagrebCroatia
  3. 3.Division of Cell Biology and BiophysicsUniversity of Missouri Kansas CityKansas CityUSA
  4. 4.Edge BioSystemsGaithersburgUSA

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