title: An improved non-denaturing method for the purification of spiralin, the main membrane lipoprotein of the pathogenic bacteria Spiroplasma melliferum.
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 170Å, 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.
authors.affiliation: UMR Université-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes Cedex, France. Electronic address: y.desfougeres@ucl.ac.uk.
authors.forename: Yann
authors.initials: Y
authors.lastname: Desfougères
authors.affiliation: Univ. Bordeaux, INRA, UMR BFP, 33882 Villenave d'Ornon Cedex, France.
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Poitou
authors.affiliation: UMR Université-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes Cedex, France.
authors.forename: Henri
authors.initials: H
authors.lastname: Wróblewski
authors.affiliation: UMR Université-CNRS 6026, Université de Rennes 1, Campus de Beaulieu, Bâtiment 13, 35042 Rennes Cedex, France; Univ. Bordeaux, INRA, UMR BFP, 33882 Villenave d'Ornon Cedex, France.
authors.forename: Laure
authors.initials: L
authors.lastname: Béven
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002793:Cholic Acids / Q000737:chemistry; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D012521:Sarcosine / Q000031:analogs & derivatives / Q000737:chemistry; D013149:Spiroplasma / Q000737:chemistry*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D002793:Cholic Acids; D003902:Detergents; C028588:spiralin; C025231:sarkosyl; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012521:Sarcosine
keywords: CE-HPLC; Detergents; Membrane lipoprotein; Mollicutes; Spiralin; Spiroplasma melliferum