Purification and characterization of BiP/Kar2 protein from Saccharomyces cerevisiae

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.