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 f...

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Bibliographic Details
Published in:Analytical biochemistry 2009-05, Vol.388 (1), p.115-121
Main Authors: Veesler, David, Blangy, Stéphanie, Siponen, Marina, Vincentelli, Renaud, Cambillau, Christian, Sciara, Giuliano
Format: Article
Language:English
Subjects:
Bacterial Proteins - chemistry
Bacterial Proteins - isolation & purification
Bacterial Proteins - metabolism
Circular Dichroism
CorA
Detergents
Detergents - chemistry
Life Sciences
Maltose - analogs & derivatives
Maltose - chemistry
Membrane Transport Proteins - chemistry
Membrane Transport Proteins - isolation & purification
Membrane Transport Proteins - metabolism
Membrane transporters
Methanosarcina - metabolism
Methanosarcina mazei
Multiangle static light scattering
Protein purification
Recombinant Proteins - chemistry
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
Spectroscopy, Fourier Transform Infrared
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Summary: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-β- 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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2009.02.018