Production and biophysical characterization of the CorA transporter from Methanosarcina mazei.
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.
Veesler
David
D
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I and II, UMR 6098, 13288 Marseille Cedex 9, France.
Blangy
Stéphanie
S
Siponen
Marina
M
Vincentelli
Renaud
R
Cambillau
Christian
C
Sciara
Giuliano
G
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017020:Methanosarcina / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared
D001426:Bacterial Proteins; D003902:Detergents; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C411362:dodecyl maltopyranoside; D008320:Maltose