Two-Step Purification of Mitochondrial Hsp70, Ssc1p, Using Mge1(His)6 Immobilized on Ni-Agarose
| Autor: | Celeste Weiss, Abdussalam Azem, Adina Niv |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Saccharomyces cerevisiae Proteins
Calcium-Transporting ATPases Saccharomyces cerevisiae Mitochondrial Membrane Transport Proteins Cofactor Fungal Proteins Sepharose Mitochondrial membrane transport protein chemistry.chemical_compound Adenosine Triphosphate ATP hydrolysis HSP70 Heat-Shock Proteins Heat-Shock Proteins Fungal protein Chromatography biology Membrane transport protein Membrane Transport Proteins Chromatography Agarose chemistry biology.protein Agarose Carrier Proteins Adenosine triphosphate Molecular Chaperones Biotechnology |
| Zdroj: | Protein Expression and Purification. 24:268-273 |
| ISSN: | 1046-5928 |
| DOI: | 10.1006/prep.2001.1563 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect