Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His) 6 immobilized on Ni-agarose

Celeste Weiss, Adina Niv, Abdussalam Azem

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)268-273
Number of pages6
JournalProtein Expression and Purification
Volume24
Issue number2
DOIs
StatePublished - Mar 2002

Fingerprint

Dive into the research topics of 'Two-step purification of mitochondrial Hsp70, Ssc1p, using Mge1(His) 6 immobilized on Ni-agarose'. Together they form a unique fingerprint.

Cite this