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

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.

Original language	English
Pages (from-to)	1344-1349
Number of pages	6
Journal	Biochemistry (Moscow)
Volume	74
Issue number	12
DOIs	https://doi.org/10.1134/S0006297909120074
Publication status	Published - Dec 2009
Externally published	Yes

Keywords

Dynamic light scattering
Expression
KCNE (MiRP)
Membrane protein
NMR
Purification

ASJC Scopus subject areas

Biochemistry

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10.1134/S0006297909120074

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Goncharuk, S. A., Shulga, A. A., Ermolyuk, Y. S., Kuzmichev, P. K., Sobol, V. A., Bocharov, E. V., Chupin, V. V., Arseniev, A. S., & Kirpichnikov, M. P. (2009). Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. Biochemistry (Moscow), 74(12), 1344-1349. https://doi.org/10.1134/S0006297909120074

Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels. / Goncharuk, S. A.; Shulga, A. A.; Ermolyuk, Ya S.; Kuzmichev, P. K.; Sobol, V. A.; Bocharov, E. V.; Chupin, V. V.; Arseniev, A. S.; Kirpichnikov, M. P.

In: Biochemistry (Moscow), Vol. 74, No. 12, 12.2009, p. 1344-1349.

Research output: Contribution to journal › Article › peer-review

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

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.",

keywords = "Dynamic light scattering, Expression, KCNE (MiRP), Membrane protein, NMR, Purification",

author = "Goncharuk, {S. A.} and Shulga, {A. A.} and Ermolyuk, {Ya S.} and Kuzmichev, {P. K.} and Sobol, {V. A.} and Bocharov, {E. V.} and Chupin, {V. V.} and Arseniev, {A. S.} and Kirpichnikov, {M. P.}",

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AU - Goncharuk, S. A.

AU - Shulga, A. A.

AU - Ermolyuk, Ya S.

AU - Kuzmichev, P. K.

AU - Sobol, V. A.

AU - Bocharov, E. V.

AU - Chupin, V. V.

AU - Arseniev, A. S.

AU - Kirpichnikov, M. P.

PY - 2009/12

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AB - 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.

KW - Dynamic light scattering

KW - Expression

KW - KCNE (MiRP)

KW - Membrane protein

KW - NMR

KW - Purification

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