TY - JOUR
T1 - Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His) 6 immobilized on Ni-agarose
AU - Weiss, Celeste
AU - Niv, Adina
AU - Azem, Abdussalam
N1 - Funding Information:
This research was supported by the Israel Science Foundation (Grant 254/99). We thank Reuven Wiener for help in carrying out the CD measurements.
PY - 2002/3
Y1 - 2002/3
N2 - 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°C.
AB - 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°C.
UR - http://www.scopus.com/inward/record.url?scp=0036514452&partnerID=8YFLogxK
U2 - 10.1006/prep.2001.1563
DO - 10.1006/prep.2001.1563
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C2 - 11858722
AN - SCOPUS:0036514452
SN - 1046-5928
VL - 24
SP - 268
EP - 273
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 2
ER -