Purification and partial molecular characterization of GRP94, an ER resident chaperone.

Journal Article (Journal Article)

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.

Full Text

Duke Authors

Cited Authors

  • Wearsch, PA; Nicchitta, CV

Published Date

  • February 1996

Published In

Volume / Issue

  • 7 / 1

Start / End Page

  • 114 - 121

PubMed ID

  • 9172775

International Standard Serial Number (ISSN)

  • 1046-5928

Digital Object Identifier (DOI)

  • 10.1006/prep.1996.0015


  • eng

Conference Location

  • United States