A procedure for the rapid purification in high yield of human glucocerebrosidase using immunoaffinity chromatography with monoclonal antibodies.
A novel chromatographic immunoaffinity procedure is described for the purification of Form I glucocerebrosidase (see J. M. F. G. Aerts, W. E. Donker-Koopman, M. K. Van der Vliet, L. M. V. Jonsson, E. I. Ginns, G. J. Murray, J. A. Barranger, J. M. Tager, and A. W. Schram, 1985, Eur. J. Biochem. 150, 565-574) from extracts of human tissues. The affinity support consists of two monoclonal anti-(glucocerebrosidase) antibodies immobilized by covalent coupling to CNBr-activated Sepharose 4B. After adsorption of the enzyme from a crude detergent extract, the column is washed successively with 30% ethylene glycol in citrate buffer (pH 6), 1% Triton X-100 in citrate phosphate buffer (pH 5.2), and 50% ethylene glycol in citrate buffer. The enzyme is eluted with 90% ethylene glycol in citrate buffer. After dilution to 30% ethylene glycol, the immunoaffinity purification is repeated. The procedure can be completed within less than 18 h. The final preparations have a high specific activity (50 U/mg protein (n = 4) for the placental enzyme) and contain no detectable impurities after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The yield is high (81 +/- 8% for the placental enzyme). The immunoaffinity column has a high capacity, can be regenerated easily, and can be utilized repeatedly without loss of activity.
D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005959:Glucosidases / Q000302:isolation & purification*; D005962:Glucosylceramidase / Q000302:isolation & purification*; D006801:Humans; D007120:Immunochemistry; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology; D010920:Placenta / Q000201:enzymology; D011247:Pregnancy; D013154:Spleen / Q000201:enzymology
D000911:Antibodies, Monoclonal; D005959:Glucosidases; D005962:Glucosylceramidase