A mild purification method for polysaccharide binding membrane proteins: phase separation of digitonin extracts to isolate the hyaluronate synthase from Streptococcus sp. in active form.
A new method was developed to purify the streptococcal hyaluronate synthase in active form to electrophoretic homogeneity. The method is based on the extraction of protoplast membranes with digitonin and a phase separation into an aqueous and a detergent phase induced by addition of polyethylene glycol 6000 at 0 degree C. Proteins bound to hyaluronate were enriched in the aqueous phase, whereas other membrane proteins resided in the detergent phase. Final purification of the hyaluronate synthase was achieved by ion exchange chromatography.
Prehm
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Institut für Physiologische Chemie und Pathobiochemie, Münster, Germany.
Nickel
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Prehm
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D000117:Acetylglucosamine / Q000032:analysis; D000595:Amino Acid Sequence; D002250:Carbon Radioisotopes; D002848:Chromatography, DEAE-Cellulose; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D003902:Detergents / Q000737:chemistry*; D004072:Digitonin / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D005079:Excipients / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D016695:Glycosyltransferases; D000076002:Hyaluronan Synthases; D007202:Indicators and Reagents / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011092:Polyethylene Glycols / Q000737:chemistry; D011134:Polysaccharides / Q000378:metabolism; D017421:Sequence Analysis; D013010:Sonication; D013291:Streptococcus / Q000201:enzymology*; D014166:Transferases; D014357:Trypsin / Q000378:metabolism; D014461:Ultracentrifugation; D029867:Xenopus Proteins
D002250:Carbon Radioisotopes; D003902:Detergents; D005079:Excipients; D007202:Indicators and Reagents; D008565:Membrane Proteins; D011134:Polysaccharides; D029867:Xenopus Proteins; D011092:Polyethylene Glycols; D014166:Transferases; D016695:Glycosyltransferases; D014453:Glucuronosyltransferase; C056944:HAS1 protein, Xenopus; D000076002:Hyaluronan Synthases; D014357:Trypsin; D004072:Digitonin; D000117:Acetylglucosamine