期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 85, 期 2, 页码 173-180出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2012.07.011
关键词
Zinc transporter; Cation diffusion facilitator; Membrane protein; Homogeneity; Mixed micelle; Maricaulis maris
类别
资金
- Irish Research Council for Science, Engineering and Technology (IRCSET)
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-beta-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. (C) 2012 Elsevier Inc. All rights reserved.
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