Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His)(6) immobilized on Ni-agarose.
The most abundant mitochondrial homolog of Hsp70, Ssc1p, is involved in the import and folding of mitochondrial proteins. We have developed an easy and efficient method for purifying Ssc1p. Following a first step of anion exchange at pH 6.6, a column of Mge1(His)(6) immobilized on Ni(2+)-agarose provides an efficient second dimension that results in highly purified protein. The strong and specific interaction between Ssc1p and its cofactor protein, Mge1, ensures that primarily functional protein is isolated. Ssc1p purified by this method hydrolyzed ATP with a turnover rate of 0.3/min. The ATP hydrolysis was enhanced slightly by Mge1, about 5 times by Mdj1, and 12 times by both cofactors together. The CD spectrum of Ssc1p had a pattern and temperature dependence similar to those shown for other hsp70 homologs, with a midpoint of the major transition at approximately 70 degrees C.
Weiss
Celeste
C
George Wise Faculty of Sciences, Department of Biochemistry, Tel Aviv University, Ramat Aviv, Israel.
Niv
Adina
A
Azem
Abdussalam
A
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification*; D002352:Carrier Proteins; D002847:Chromatography, Agarose / Q000379:methods*; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins / Q000302:isolation & purification; D006360:Heat-Shock Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose
D002352:Carrier Proteins; D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C086039:MGE1 protein, S cerevisiae; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D018832:Molecular Chaperones; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D012685:Sepharose; D000252:Calcium-Transporting ATPases