Maricaulis maris cation diffusion facilitator: Achieving homogeneity through a mixed-micelle approach

David Russell, Olga Kolaj-Robin, Tewfik Soulimane

Research output: Contribution to journalArticlepeer-review

Abstract

One of the most common problems encountered during isolation and purification of homogenous membrane proteins is their aggregation which in turn makes the obtained material unsuitable for structural and functional studies. As various detergents can have a different impact on the protein stability and solubility, introducing the protein to different detergent micelles can result in the removal of such aggregation. Here we describe a method for retrieving homogenous samples of a putative member of the cation diffusion facilitator family from the marine bacterium Maricaulis maris (MmCDF3). A feature that makes this 23 kDa protein particularly interesting to study is that it lacks the cytoplasmic domain that in other members of the CDF family protrudes into the cytoplasm and that was proposed to play a crucial role in the metal transport. The MmCDF3 was produced with the C-terminal hexahistidine tag in Escherichia coli and subsequently purified using affinity chromatography followed by gel-filtration yielding 7.5 mg of the pure transporter. However, solubilization and purification of the protein in a single detergent or a complete detergent exchange to another single detergent invariably resulted in the formation of protein aggregates. Instead, if the protein was introduced into a micelle of multiple detergents, the aggregation level notably decreased. Purification of the protein in a mixture of n-dodecyl-β-D-maltoside and Fos-choline-12 at a ratio of 4 to 1 allowed for recovery of 3.7 mg of homogenous, non-aggregated MmCDF3 from 1 L of bacterial culture that could easily be separated from aggregated material.

Original languageEnglish
Pages (from-to)173-180
Number of pages8
JournalProtein Expression and Purification
Volume85
Issue number2
DOIs
Publication statusPublished - 1 Oct 2012

Keywords

  • Cation diffusion facilitator
  • Homogeneity
  • Maricaulis maris
  • Membrane protein
  • Mixed micelle
  • Zinc transporter

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