Bacterial synthesis, purification, and solubilization of membrane protein KCNE3, a regulator of voltage-gated potassium channels
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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 wordsKCNE (MiRP) membrane protein expression purification dynamic light scattering NMR
histidine tag (HHHHHH)
Heteronuclear Single Quantum Correlation
immobilized metal affinity chromatography
flexible linker (GSGSG)
thrombin recognition site (LVPRGS)
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- 13.Kirpichnikov, M. P., Goncharuk, M. V., Ermolyuk, Ya. S., Goncharuk, S. A., Shulga, A. A., Maslennikov, I. V., and Arseniev, A. S. (2005) Technol. Live Syst., 2, 20–27.Google Scholar