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Biochemistry (Moscow)

, Volume 74, Issue 12, pp 1344–1349 | Cite as

Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels

  • S. A. GoncharukEmail author
  • A. A. Shulga
  • Ya. S. Ermolyuk
  • P. K. Kuzmichev
  • V. A. Sobol
  • E. V. Bocharov
  • V. V. Chupin
  • A. S. Arseniev
  • M. P. Kirpichnikov
Article

Abstract

An efficient method is described for production of membrane protein KCNE3 and its isotope labeled derivatives (15N-, 15N-/13C-) in amounts sufficient for structural-functional investigations. The purified protein preparation within different detergent micelles was characterized using dynamic light scattering, CD spectroscopy, and NMR spectroscopy. It is shown that within DPC/LDAO micelles the protein is in monomeric form and acquires mainly α-helical conformation. The existence of cross-peaks for all glycines of the 15N-HSQC NMR spectra as well as relatively small line widths (∼20 Hz) confirm the high quality of the preparation and the possibility of obtaining structural-dynamic information on KCNE3 by high resolution heteronuclear NMR spectroscopy.

Key words

KCNE (MiRP) membrane protein expression purification dynamic light scattering NMR 

Abbreviations

DPC

dodecyl phosphocholine

H6

histidine tag (HHHHHH)

HSQC

Heteronuclear Single Quantum Correlation

IMAC

immobilized metal affinity chromatography

IPTG

isopropyl β-D-thiogalactoside

GS

flexible linker (GSGSG)

LDAO

lauryl dimethylaminoxide

LMPC

lysomyristoyl phosphatidylcholine

LMPG

lysomyristoyl phosphatidylglycerol

LPPG

lysopalmitoyl phosphatidylglycerol

TR

thrombin recognition site (LVPRGS)

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Copyright information

© Pleiades Publishing, Ltd. 2009

Authors and Affiliations

  • S. A. Goncharuk
    • 1
    Email author
  • A. A. Shulga
    • 1
  • Ya. S. Ermolyuk
    • 1
  • P. K. Kuzmichev
    • 1
  • V. A. Sobol
    • 1
  • E. V. Bocharov
    • 1
  • V. V. Chupin
    • 1
  • A. S. Arseniev
    • 1
  • M. P. Kirpichnikov
    • 1
  1. 1.Shemyakin and Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia

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