Purification and characterization of the major lipoprotein (P28) of Spiroplasma apis.
The plasma membrane of Spiroplasma apis contains a 28-kDa major protein (P28), like other spiroplasmas which also possess a main 26- to 28-kDa membrane polypeptide, called spiralin. In the work described here, we have developed a simple and efficient method for the purification of P28 of this mollicute, a wall-less eubacteria. Proteins were first selectively extracted from the isolated membrane with the mild detergents (i) sodium N-lauroylsarcosinate (Sarkosyl) and (ii) 3-[(3-cholamidopropyl)dimethylamonio]-1-propyl sulfonate (Chaps) and subjected to size-exclusion HPLC in the presence of Chaps. The P28-enriched fraction was thereafter subjected to the second chromatographic step involving cation exchange HPLC in the presence of the same detergent. P28 was purified at the milligram level (yield, 40%). Metabolite labeling with [14C]palmitic acid and chemical analysis of P28 indicated that it is covalently modified by two O-ester-bound fatty acids and one amide-linked chain and contains a S-glycerylcysteine at the N-terminus. By charge-shift electrophoresis, Triton X-114 phase separation, and growth inhibition tests it was shown that P28 is a typical amphiphilic protein exposed, at least partly, at the cell surface. Together, our data provided evidence that P28 is a "classical" lipoprotein (i.e., triacylated) like the members of the spiralin family.
Le Hénaff
Michel
M
Groupe "Membranes et Osmorégulation", Université de Rennes 1, F-35042 Rennes Cedex, France. m-lehenaff@enitab.fr
Crémet
Jean-Yves
JY
Fontenelle
Catherine
C
D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D008074:Lipoproteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D013149:Spiroplasma / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins