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

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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.

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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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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    S. A. Goncharuk, A. A. Shulga, Ya. S. Ermolyuk, P. K. Kuzmichev, V. A. Sobol, E. V. Bocharov, V. V. Chupin, A. S. Arseniev & M. P. Kirpichnikov

Authors
  1. S. A. Goncharuk
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  2. A. A. Shulga
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  3. Ya. S. Ermolyuk
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  4. P. K. Kuzmichev
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  5. V. A. Sobol
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  6. E. V. Bocharov
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  7. V. V. Chupin
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  8. A. S. Arseniev
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  9. M. P. Kirpichnikov
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Correspondence to S. A. Goncharuk.

Additional information

Original Russian Text © S. A. Goncharuk, A. A. Shulga, Ya. S. Ermolyuk, P. K. Kuzmichev, V. A. Sobol, E. V. Bocharov, V. V. Chupin, A. S. Arseniev, M. P. Kirpichnikov, 2009, published in Biokhimiya, 2009, Vol. 74, No. 12, pp. 1650–1656.

Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM09-124, September 27, 2009.

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Goncharuk, S.A., Shulga, A.A., Ermolyuk, Y.S. et al. Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. Biochemistry Moscow 74, 1344–1349 (2009). https://doi.org/10.1134/S0006297909120074

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  • DOI: https://doi.org/10.1134/S0006297909120074

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