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.
Tokunaga
M
M
Laboratory of Molecular Genetics, Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kawamura
A
A
Kohno
K
K
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002845:Chromatography; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D017886:Durapatite; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005800:Genes, Fungal / Q000187:drug effects; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D006882:Hydroxyapatites; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D000516:alpha-Amylases / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006882:Hydroxyapatites; C060153:KAR2 protein, yeast; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; D017886:Durapatite; D000516:alpha-Amylases; D000251:Adenosine Triphosphatases