Epitope tagging of the human endoplasmic reticulum HSP70 protein, BiP, to facilitate analysis of BiP--substrate interactions

Anal Biochem. 1995 Aug 10;229(2):170-9. doi: 10.1006/abio.1995.1399.

Abstract

We modified BiP, the resident endoplasmic reticulum (ER) heat shock protein 70, to contain an epitopetag sequence close to the C-terminus (BiP-tag); the epitope is derived from an influenza hemagglutinin (HA) subtype and is recognized by the monoclonal antibody 12CA5. This antibody both immunoprecipitates BiP-tag and detects it on Western blots. Using transient expression of cDNAs in COS cells, we studied the interaction of BiP-tag with several membrane proteins. Consistent with previous work on BiP, BiP-tag bound poorly and transiently to newly made wild-type influenza HA glycoprotein and strongly and irreversibly to an HA mutant that misfolds and is retained in the ER. Most newly made erythropoietin receptor (EPO-R) polypeptides are retained in the ER and degraded there; we show here that, in cotransfected COS cells, newly made EPO-R is bound to BiP-tag prior to its degradation. Thus, by several criteria the BiP-tag molecule is fully functional in binding newly made proteins. Because it can be immunoprecipitated by a readily available antibody, it offers several advantages to the study of protein folding in the ER and the role of chaperones in this process.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism*
  • Cell Line
  • Chlorocebus aethiops
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • Endoplasmic Reticulum / metabolism*
  • Epitopes / analysis*
  • HSP70 Heat-Shock Proteins / biosynthesis
  • HSP70 Heat-Shock Proteins / isolation & purification
  • HSP70 Heat-Shock Proteins / metabolism*
  • Heat-Shock Proteins*
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Hemagglutinins, Viral / metabolism
  • Humans
  • Kidney
  • Molecular Chaperones / biosynthesis
  • Molecular Chaperones / isolation & purification
  • Molecular Chaperones / metabolism*
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Protein Binding
  • Receptors, Erythropoietin / isolation & purification
  • Receptors, Erythropoietin / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Tagged Sites
  • Transfection

Substances

  • Carrier Proteins
  • DNA Primers
  • Epitopes
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Hemagglutinins, Viral
  • Molecular Chaperones
  • Receptors, Erythropoietin
  • Recombinant Proteins
  • molecular chaperone GRP78