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GST-tagged Yeast Protein Purification

HI Hogune Im

Published: Oct 5, 2011 DOI: 10.21769/BioProtoc.141 Views: 16904

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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

  1. Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (Sigma-Aldrich, catalog number: E0396 )
  2. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  3. HEPES (Sigma-Aldrich, catalog number: 54457 )
  4. Roche protease inhibitor tablets (containing EDTA) (Roche Diagnostics, catalog number: 11697498001 )
  5. Phosphatase inhibitor (Roche Diagnostics, catalog number: 4906845001 )
  6. Glutathione (Axxora, catalog number: 157-002-G005 )
  7. Galactose (Mp Biomedicals, catalog number: 0210174701 )
  8. Zirconia/Silica beads (Biospec Products, catalog number: 11079105z )
  9. Glutathione beads (Thermo Fisher Scientific, catalog number: 16100 )
  10. Tris
  11. NaCl
  12. TritonX-100
  13. Glycerol
  14. Beta-mercaptoethanol (BME)
  15. Sc-ura liquid media with raffinose
  16. Lysis buffer (see Recipes)
  17. Wash buffer I (see Recipes)
  18. Wash buffer II (see Recipes)
  19. Elution buffer (see Recipes)

Equipment

  1. Table top centrifuge
  2. Bead beater
  3. 50 ml Falcon tubes

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Biochemistry > Protein > Isolation and purification

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2 Q&A

May I ask you the recipe for preparing sc-ura/raffinose?...
edit 0 Answer 1 View Jul 13, 2012

I have a question. What exactly is a bead beater? And where...
edit 1 Answer 3 Views Jan 31, 2012