Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae

J Biol Chem. 1992 Sep 5;267(25):17553-9.

Abstract

Using specific anti-BiP/Kar2 antibody as the probe, we have developed an efficient purification method of BiP/Kar2 protein from the total cell extract of Saccharomyces cerevisiae. Overproduction of BiP/Kar2 protein was achieved by the cloning of the KAR2 gene on multicopy plasmids and the treatment of cells harboring the cloned KAR2 gene with tunicamycin. Freeze-thaw treatment, hydroxyapatite high pressure liquid chromatography, and ATP-agarose column chromatography of crude extract yielded homogeneous BiP/Kar2 protein (including less than 0.2% of degradative derivative) with a 430-fold purification and 28% recovery. Edman degradation of purified BiP/Kar2 suggests that the mature protein corresponds to a processed product with the removal of a 42-amino acid presequence. It is active as a homodimer and exhibits ATPase activity with a specific activity of 2 pmol/min/micrograms of protein. Protease susceptibility indicated that the ADP form of BiP/Kar2 is more resistant than the ATP form to the chymotrypsin digestion and that BiP/Kar2 required the presence of ATP to avoid the irreversible denaturation. Synthesis of BiP/Kar2 was induced by the inducible expression of an aberrant heterologous protein, yeast killer prepro-signal mouse alpha-amylase fusion protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / isolation & purification
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Sequence
  • Animals
  • Antibodies
  • Chromatography
  • Chromatography, Affinity
  • Cloning, Molecular
  • Durapatite
  • Fungal Proteins / genetics
  • Fungal Proteins / isolation & purification*
  • Fungal Proteins / metabolism
  • Genes, Fungal / drug effects
  • HSP70 Heat-Shock Proteins*
  • Heat-Shock Proteins / isolation & purification*
  • Hydroxyapatites
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Tunicamycin / pharmacology
  • alpha-Amylases / isolation & purification
  • alpha-Amylases / metabolism

Substances

  • Antibodies
  • Fungal Proteins
  • HSP70 Heat-Shock Proteins
  • Heat-Shock Proteins
  • Hydroxyapatites
  • KAR2 protein, yeast
  • Recombinant Fusion Proteins
  • Tunicamycin
  • Durapatite
  • alpha-Amylases
  • Adenosine Triphosphatases