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381.
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. 相似文献
382.
383.
A crude glucosyltransferase (GTase) preparation was obtained from the culture supernatant of Streptococcus mutans strain MT703 (serotype e) by 50% ammonium sulphate precipitation. Antiserum specific against the GTase was prepared by immunizing rabbits intramuscularly with the GTase in Freund incomplete adjuvant, followed by GTase without adjuvant intravenously. Gamma globulin fractions of the antiserum and normal serum were partially purified by 1/3 saturated ammonium sulphate precipitation. The antibody strongly inhibited the GTase activity (greater than 90%) of type c, e and f S. mutans, whereas the GTase of type a, d and g was not affected by the antibody. The GTase from type b S. mutans was slightly inhibited. The adherence of viable cells of type c, e, and f S. mutans to a glass surface due to synthesis of glucan by the cell-associated GTase was also significantly inhibited by the antibody to the enzyme. These results suggest that type c, e, and f and types a, d, and g S. mutans can be separated into two major groups in terms of the immunological relationship of GTase. 相似文献
384.
Mass vaccination campaigns against viral diseases, both human and animal, depend on the availability of cheap viral antigens. The eukaryote Dictyostelium discoideum has simple growth requirements and rapid growth rates and forms stable cell lines. These features, together with the possibility of secreting recombinant (glyco) proteins into a defined buffer, make the D. discoideum expression system an attractive option for producing economical recombinant subunit vaccines. 相似文献