Modified cell ELISA to determine the solubilization of cell surface proteins: Applications in GPI-anchored protein purification.
A major step in purifying membrane bound proteins involves the solubilization of the protein of interest from the cell membranes. Glycosylphosphatidyl inositol (GPI)-anchored proteins pose a singular problem in this solubilization step since they are found in detergent-resistant membrane complexes and accordingly are insoluble in cold Triton X-100. In this study we have developed a modified cell ELISA that determines the solubility of these cell surface proteins under various solubilization conditions. Using this non-radioactive method we show that the combination of saponin/Triton X-100 at 4 degrees C solubilized GPI-anchored proteins more efficiently than Triton X-100 at 4 degrees C. The combination of saponin/Triton X-100 at 4 degrees C avoids the potential of activating proteases that occurs when using Triton X-100 at 37 degrees C. Furthermore, our method also shows the saponin/Triton X-100 solubilized GPI-anchored proteins equivalent to the more expensive octyl beta-glucoside. This is a particularly important consideration in large-scale protein purification. This method obviates the need to use radioactivity, gel electrophoresis and immunoblotting procedures. The solubilization conditions determined by this modified ELISA are readily translated to the practical application of large-scale protein purification as demonstrated in the purification of two different recombinant GPI-anchored proteins, GPI-hB7-1 (CD80) and GPI-mICAM-1 (CD54).
Department of Pharmaceutical Sciences, Mercer University School of Pharmacy, Atlanta, GA 30341, USA.
Gary W
GW
Bumgarner
Jamie C
JC
Zampell
Shanmugam
S
Nagarajan
Neil J
NJ
Poloso
Amanda S
AS
Dorn
Martin J
MJ
D'Souza
Periasamy
P
Selvaraj
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis; D018122:B7-1 Antigen / Q000096:biosynthesis / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005434:Flow Cytometry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry*; D006825:Hybridomas / Q000378:metabolism; D015151:Immunoblotting; D046911:Macromolecular Substances / Q000737:chemistry; D017830:Octoxynol / Q000494:pharmacology; D017452:Receptors, IgG / Q000096:biosynthesis; D012995:Solubility; D013696:Temperature; D014162:Transfection
D015703:Antigens, CD; D018122:B7-1 Antigen; D003902:Detergents; C080568:Fc gamma receptor IIA; D017261:Glycosylphosphatidylinositols; D046911:Macromolecular Substances; D017452:Receptors, IgG; D017830:Octoxynol