Purification of a membrane glycoprotein with an inositol-containing phospholipid anchor from Dictyostelium discoideum.
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.
School of Biological Sciences, Macquarie University, Sydney, Australia.
Z C
ZC
Ti
A A
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Gooley
M B
MB
Slade
V M
VM
Bowers
K L
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Williams
D000911:Antibodies, Monoclonal; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D003902:Detergents; D004023:Dictyostelium / Q000032:analysis* / Q000254:growth & development; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006017:Glycolipids / Q000502:physiology*; D017261:Glycosylphosphatidylinositols; D007118:Immunoassay; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D010716:Phosphatidylinositols / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013863:Thioglucosides
D000911:Antibodies, Monoclonal; D003902:Detergents; D005656:Fungal Proteins; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D010716:Phosphatidylinositols; D011994:Recombinant Proteins; D013863:Thioglucosides; C042989:n-octyl-beta-D-thioglucopyranoside