Affinity purification of molecular chaperones of the yeast Hansenula polymorpha using immobilized denatured alcohol oxidase.
We used peroxisomal alcohol oxidase (AO) for the affinity purification of molecular chaperones from yeasts. Methodical studies showed that up to 0.8 mg of purified bacterial GroEL was able to bind per ml of immobilized denatured AO column material. Using crude extracts of Hansenula polymorpha or Saccharomyces cerevisiae, several proteins were specifically eluted with Mg-ATP which were recognized by antibodies against hsp60 or hsp70. One H. polymorpha 70 kDa protein was strongly induced during growth at elevated temperatures, whereas a second 70 kDa protein as well as a 60 kDa protein showed strong protein sequence homology to mitochondrial SSC1 and hsp60, respectively, from S. cerevisiae.
Evers
M E
ME
Laboratory for Electron Microscopy, University of Groningen, Haren, The Netherlands.
Huhse
B
B
Titorenko
V I
VI
Kunau
W H
WH
Hartl
F U
FU
Harder
W
W
Veenhuis
M
M
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D018834:Chaperonin 60; D018833:Chaperonins; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized; D005656:Fungal Proteins / Q000302:isolation & purification; D006360:Heat-Shock Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000737:chemistry*; D011489:Protein Denaturation; D011506:Proteins / Q000302:isolation & purification*
D001426:Bacterial Proteins; D018834:Chaperonin 60; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D006360:Heat-Shock Proteins; D011506:Proteins; D000429:Alcohol Oxidoreductases; C019643:alcohol oxidase; D018833:Chaperonins