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RESEARCH DIVISION Laboratory Manual

 


 

Purification of GST Fused Proteins

  Day 1
  1. Set up an overnight culture in 100 ml LMM broth or 100 ml terrific broth containing 100ul 100 mg/mlAmp
  Day 2
  1. Add 40-50 ml o/n culture to 1 lt terrific/K2K (see appendix) with 0.5 ml 100mg/ml Amp.
  2. Grow at 37C to OD600 of approximately 0.5-0.8 (~90-150 min)
  3. Induce with 0.5ml 50mg/ml 1PTG prepared freshly in DDW.
  4. Incubate 2-3 hrs 37C for stable proteins, 1 hr 37C for unstable proteins (room temperature expression can be tried for some insoluble proteins, but the medium should be cooled before induction)
  5. Pellet cells in GS3 rotor 5000rpm 10 min at 4C.
  6. Pool cells in small amount of medium and then pellet GSA rotor 5000 rpm 10 min, 4C and remove all supernatant.
  7. This is a convenient point to stop and to store pellets at -70C.
  Day 3
  1. Place GSA tube on ice
  2. Resuspend pellet in 40 ml of ice cold PBS/10 mM DTT/1mM PMSF (see reagents for PMSF stock) / 0.2 mg/ml lysozyme (in DDW)
  3. Incubate ice 30 min
  4. Add 4.5 ml 10% Triton X-100, swirl
  5. Add 450ul 1M MgCl2, swirl (final concentration 10mM Mg2+)
  6. Add 450ul 10mg/ml DNasel (Boehringer #1284 932), swirl (final concentration 100ug/ml)
  7. Incubate ice 30 min. Start step 8. immediately.
  8. Preswell glutathione agarose beadsby adding 75mg beads to 10ml tube (#G-4510, 12 spacer atoms Sigma) Add PBS/10mM DTT/1%TX-100 and place on wheel for 30 min. Wash 2X in PBS/10mM DTT/1%TX-100. Alternatively, use pre-swollen Pharmacia Glutathione Sepharose beads. These can be washed rapidly 3 times in PBS/10mM DTT/1%TX100.
  9. Pellet debris 20 min in a prechilled SS34 at 15000 rpm
  10. Add supernatant to 1 ml preswollen glutathione beads in a 50 ml tube and place on wheel at 4C for 1 hr
  11. Pellet beads in a benchtop centrifuge at 2K for 1-2min at 4C. Allow centrifuge to slow down without the brake (the beads are easily resuspended if the centrifuge slows too fast).
  12. Aspirate the supernatant and then add 50ml ice cold PBS/10mM DTT/1% TX-100. Swirl and repeat step 11 twice (three washes).
  13. Add 5ml PBS/10mM DTT/1% TX-100 and transfer to a 10ml tube by pouring (beads tend to stick to a pipette). Rinse the tube and pool remaining beads. Remove all overlying supernatant.
  14. Prepare ice cold 5mM Glutathione (Sigma G-4251, reduced form), 50mM Tris pH 8.0 freshly and add 1ml to beads to elute fusion protein. Incubate for 5' on ice.
  15. Pellet as in step 11 and remove the remove supernatant
  16. Repeat twice more. Some recommend using pH9.6 Tris to shift some proteins off the beads but we have not found that necessary.
  17. Run aliquots on 10-12% protein gel (Gex is 26K plus predicted size of fused protein). Use 5ul eluate in 20ul SDS sample buffer (boiled) and appropriate molecular weight markers. For stable fused proteins a thick (2-5mm) band should be obtained with the first eluate, and about half to third as much in the second.
  18. Pool fractions containing a significant amount of protein and then dialyse with 1000 X volume of eluate of ice cold PBS or borate or MOPS. 0.1M borate, pH 8.8 is used for proteins to be biotinylated. Dialyse at 4C for 24-48 hrs with one change.
  19. Check the recovery of dialysed protein on gel and freeze aliquots at -70C
  20.  

  Reuse of beads:
  1. Rinse with: 3M NaCl to remove all bound protein, then rinse with PBS/10mM DTT/1% TX100 3 times
  2. Store in: PBS/10mM DTT/1% TX-100
  Notes:
  1. Only use preused beads for preparing the same protein that the beads were used for previously, as cleaning of the beads is not 100% effective.
  2. Protein Estimation using the BioRad Reagent (#500-0006)
  3. Setup sufficient ependorf tubes for a standard curve with 0, 5, 10, 15, 20, 25ug of BSA and the samples to be tested.
  4. Add 200ul of BioRad reagent and 800ul DDW.
  5. Add samples (5ul of eluate) or BSA controls to tubes and vortex immediately. Concentrated samples may aggregate if not dispersed immediately.
  6. Read OD595 and generate standard curve to read off unknowns.
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998