Purification and partial molecular characterization of GRP94, an ER resident chaperone.
GRP94 is a resident glycoprotein of the endoplasmic reticulum (ER) and a member of the hsp90 family of molecular chaperones. Current experimental evidence indicates that GRP94 functions in an as yet undefined manner in protein folding and assembly in the ER. We report a rapid, high-yield GRP94 purification procedure that yields milligram quantities of homogeneous protein suitable for structural and biochemical analyses. Beginning with a rough microsome fraction derived from porcine pancreas, GRP94 was isolated by partial detergent extraction, anion exchange, and gel filtration chromatography. With this procedure, approximately 3 mg of homogeneous GRP94 can be prepared from a 25-g pancreas. Heterogeneity in the migration of purified GRP94 on native and denaturing PAGE was observed and demonstrated to reflect variability in the N-linked glycosylation state of the protein. Analysis by native and two-dimensional nonreducing/reducing gels indicated that the protein exists as a dimer of noncovalently associated subunits. The membrane localization of GRP94 in isolated pancreatic microsomes was assessed by alkali and detergent extraction. By comparison with the resident ER integral membrane protein TRAPalpha, GRP94 exists as a soluble, lumenal protein.
Wearsch
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Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Nicchitta
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D000818:Animals; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D018870:Endoplasmic Reticulum, Rough / Q000737:chemistry*; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification*; D006596:Hexosaminidases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D018832:Molecular Chaperones / Q000737:chemistry / Q000302:isolation & purification*; D010179:Pancreas / Q000737:chemistry; D013552:Swine
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; C018551:glucose-regulated proteins; D006596:Hexosaminidases; D004229:Dithiothreitol