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GST-tagged Yeast Protein Purification
Published: Oct 5, 2011 DOI: 10.21769/BioProtoc.141 Views: 16930
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Abstract
Glutation S-transferase (GST) tagging is the most commonly used purification strategy for recombinant protein. It was developed with the goal of preserving the enzymatic activity by utilizing gentle elution condition of the target protein from purification matrix (Poon and Hunt., 1994). The method described here can be applied from single protein to proteome scale purification of recombinant protein from yeast (Zhu et al., 2000; Zhu et al., 2001).
Materials and Reagents
- Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E0396 )
- Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
- HEPES (Sigma-Aldrich, catalog number: 54457 )
- Roche protease inhibitor tablets (containing EDTA) (Roche Diagnostics, catalog number: 11697498001 )
- Phosphatase inhibitor (Roche Diagnostics, catalog number: 4906845001 )
- Glutathione (Axxora, catalog number: 157-002-G005 )
- Galactose (Mp Biomedicals, catalog number: 0210174701 )
- Zirconia/Silica beads (Biospec Products, catalog number: 11079105z )
- Glutathione beads (Thermo Fisher Scientific, catalog number: 16100 )
- Tris
- NaCl
- TritonX-100
- Glycerol
- Beta-mercaptoethanol (BME)
- Sc-ura liquid media with raffinose
- Lysis buffer (see Recipes)
- Wash buffer I (see Recipes)
- Wash buffer II (see Recipes)
- Elution buffer (see Recipes)
Equipment
- Table top centrifuge
- Bead beater
- 50 ml Falcon tubes
Procedure
Category
Biochemistry > Protein > Isolation and purification
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