RESUMEN
Large-scale purification of a Dictyostelium discoideum cell surface glycoprotein, which is anchored in the membrane via a glycosylphosphatidylinositol (GPI) moiety, is described. The purification protocol involved four steps: separation of crude cell membranes by low-speed centrifugation, delipidization of these membranes using acetone, extraction of the membrane proteins using the detergent Octyl beta-D-thioglucopyranoside (OTP), and purification of a specific membrane protein by monoclonal antibody immunoaffinity chromatography. The protein purified, PsA (prespore-specific antigen), is a developmentally regulated membrane glycoprotein found on a subset of cells from the cellular slime mould, D. discoideum. The protocol provides an efficient, economical, and technically simple way to purify GPI proteins in sufficient quantities for structural and functional studies. PsA was recovered at a yield of about 60%; with a purity of 97%, the extraction of 1 x 10(10) cells (1.1 g dry weight) yielded about 0.5 mg PsA glycoprotein. Techniques are described for growing kilogram quantities of D. discoideum cells in stainless steel trays at little cost. D. discoideum has considerable potential as a novel expression system for the production of foreign membrane-associated proteins. The purification strategy provides a means of purifying other GPI proteins, including those produced by protein engineering techniques.
Asunto(s)
Dictyostelium/análisis , Proteínas Fúngicas/aislamiento & purificación , Glucolípidos/fisiología , Glicoproteínas de Membrana/aislamiento & purificación , Fosfatidilinositoles/fisiología , Anticuerpos Monoclonales , Fraccionamiento Celular , Membrana Celular/química , Cromatografía de Afinidad , Detergentes , Dictyostelium/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Proteínas Fúngicas/química , Glicosilfosfatidilinositoles , Inmunoensayo , Glicoproteínas de Membrana/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , TioglucósidosRESUMEN
Cellulose is one of the commonest structural biopolymers. How cellulose is organized in extracellular matrices is a mystery. Here we investigate a model system, the extracellular matrix (ECM) of Dictyostelium discoideum which is composed of proteins and cellulose. A group of glycoproteins, the sheathins, which colocalize with cellulose in the ECM of D. discoideum are characterized. Sheathins are dimeric or trimeric forms of molecular mass 53-68 kDa, where the monomers are 12-35 kDa. The sheathin subunits are similar but not identical proteins. The sheathin family comprises sheathin 68, (68-kDa trimer); sheathin 62, (62-kDa dimer); sheathin 55, (55-kDa dimer), and sheathin 53, (53-kDa dimer). The subunits which assemble into the four sheathins represent at least three gene products: ShC, ShD, and ShE which are linked by disulphide bonds. Protein sequence analysis shows two of the sheathin genes encode products ShC and ShD with very similar amino terminal sequences. This group of D. discoideum ECM glycoproteins has homology with two other much larger ECM proteins of D. discoideum, ST430 and ST310, which are located in a more dispersed fashion in the ECM. Sheathins are tightly but non-covalently associated with the ECM, and this association requires strong denaturing conditions for disruption, e.g., SDS or 8 M urea. Sheathins form a component of the "cell prints" which are believed to have a role in cell-ECM interactions and slug cell migration.