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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
Article

Abstract

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.

Keywords

Chaperonin Osmolytes Affinity tags Protein folding Protein stability 

Abbreviations

IPTG

Isopropyl-β-d-thiogalactopyranoside

Ni-NTA

Nickel-nitrilotriacetic acid

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SUMO

Small ubiquitin-like modifier

DTT

Dithiothreitol

PKD1

Polycystin-1

PKD2

Polycystin-2

PEPCK

Phosphoenolpyruvate carboxykinase

GST

Glutathione-S-transferase

Notes

Acknowledgments

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.

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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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