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Production and biophysical characterization of the CorA transporter from Methanosarcina mazei.

Abstract : We report here a general strategy to overproduce and characterize membrane transporters. To illustrate our approach, we selected one member of the CorA transporter family among four tested that belonged to different species. This approach is transposable to other membrane proteins and involves the following steps: (i) cloning by homologous recombination, (ii) high-throughput expression screening, (iii) fermenter-based large-scale production, (iv) high-throughput detergent solubilization screening, (v) protein purification, (vi) multiangle static light scattering/refractometry characterization of purified proteins, (vii) circular dichroism spectroscopy, and (viii) detergent concentration measurements by Fourier transform infrared (FT-IR) spectroscopy. Methanosarcina mazei CorA was expressed in milligram quantities and purified (> 95% pure). n-Dodecyl-beta-D-maltopyranoside (DDM) retained the pentameric native structure of this transporter; thus, we selected it as working detergent. Furthermore, we measured the detergent concentration in our purified and concentrated protein sample by FT-IR to maintain it as low as possible. Our strategy can be adapted to many structural biology approaches as well as for study of single membrane proteins in a variety of conditions.
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David Veesler, Stéphanie Blangy, Marina Siponen, Renaud Vincentelli, Christian Cambillau, et al.. Production and biophysical characterization of the CorA transporter from Methanosarcina mazei.. Analytical Biochemistry, Elsevier Masson, 2009, 388 (1), pp.115-21. ⟨10.1016/j.ab.2009.02.018⟩. ⟨hal-01615932⟩



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