Desfougeres, Y;
Poitou, J-M;
Wroblewski, H;
Beven, L;
(2016)
An improved non-denaturing method for the purification of spiralin, the main membrane lipoprotein of the pathogenic bacteria Spiroplasma melliferum.
Journal of Chromatography B
, 1036-7
pp. 149-156.
10.1016/j.jchromb.2016.10.012.
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Abstract
Spiralin is the most abundant protein of several species of spiroplasmas, helical, motile bacteria pathogenic for arthropods and plants. This amphiphilic protein is anchored to the outer face of the plasma membrane by a lipoylated N-terminal cysteine. Although spiroplasma pathogenicity in mammals is controversial, it was shown that spiralin is highly immunogenic and endowed with immunomodulatory activity. In this paper, we describe a high performance method for the purification of Spiroplasma melliferum spiralin under non-denaturing conditions. The protein was selectively extracted with 3-[(3-cholamidopropyl) dimethylammonio]-1-propyl sulfonate (CHAPS) from the membrane pre-treated with sodium dodecyl-N-sarcosinate (Sarkosyl), and purified to homogeneity by cation-exchange HPLC with an overall yield of ∼60%. Detergent-depleted, water-soluble micelles of spiralin displaying a mean diameter of 170 Å, as evidenced by transmission electron microscopy, were obtained by dialysis detergent removal. Circular dichroism spectroscopy and cross immunoprecipitation assay of the purified spiralin strongly suggested that this purification method could retain the structural characteristics of the native spiralin. The strategy developed to purify spiralin (two successive selective extractions of membrane proteins with mild detergents followed by ion-exchange chromatography) should prove useful for the purification of membrane lipoproteins of other bacteria of the class Mollicutes including different pathogens for humans, animals and plants.
| Type: | Article |
|---|---|
| Title: | An improved non-denaturing method for the purification of spiralin, the main membrane lipoprotein of the pathogenic bacteria Spiroplasma melliferum |
| Open access status: | An open access version is available from UCL Discovery |
| DOI: | 10.1016/j.jchromb.2016.10.012 |
| Publisher version: | http://dx.doi.org/10.1016/j.jchromb.2016.10.012 |
| Language: | English |
| Additional information: | Copyright © 2016. This manuscript version is published under a Creative Commons Attribution Non-commercial Non-derivative 4.0 International licence (CC BY-NC-ND 4.0). This licence allows you to share, copy, distribute and transmit the work for personal and non-commercial use providing author and publisher attribution is clearly stated. Further details about CC BY licences are available at http://creativecommons.org/licenses/by/4.0. Access may be initially restricted by the publisher. |
| Keywords: | CE-HPLC; Detergents; Membrane lipoprotein; Spiralin; Spiroplasma melliferum; Mollicutes |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Lab for Molecular Cell Bio MRC-UCL |
| URI: | https://discovery-pp.ucl.ac.uk/id/eprint/1524082 |
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