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Bacterial Synthesis, Purification, and Solubilization of Membrane Protein KCNE3, a Regulator of Voltage-Gated Potassium Channels

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

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; fax: (495) 335-5033; E-mail: goncharuk@nmr.ru

* To whom correspondence should be addressed.

Received April 17, 2009; Revision received June 5, 2009
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

DOI: 10.1134/S0006297909120074