Difference between revisions of "Purification of MBP Fusion Proteins"

Latest revision as of 22:00, 2 December 2010

Bacteria Production and Induction

• Express and induce protein in culture under appropriate conditions:

1. grow an overnight culture in ~25 mL LB/Amp (with Chloramphenicol if using Rosetta cells) from a colony <2 weeks post transformation.
2. add 5 mL overnight culture to 1L TB/Amp and grow at 37C
3. grow to OD600 of 0.6-1.0 and induce with 100 uM IPTG (the concentration of IPTG and duration of induction should be optimized for each protein)
4. let grow O/N at <25C (optimize induction time/temp for each protein, see Induction Conditions).
5. centrifuge cells 10 min at 9000 RPM to pellet bacteria freeze if stopping at this point.

Lysis and Purification

1. Resuspend cells in ~20 mL lysis buffer (PBS + 0.1% B-ME and a PI tablet). Freeze in liquid nitrogen if not continuing with purification
2. French Press cells 2 x 15 000 psi (see French Press Protocol)
3. Centrifuge lysate at >15 000 RPM (i.e. 20,000RMP in JA25.5 at 4 degrees C) for 30 min to clarify
4. Add 1.0 mL amylose resin to 50 mL PBS (no PI) to prepare beads. Let sit for 30 min. to allow beads to settle or pellet beads for 5 min at 1000 RPM and remove buffer
5. Save a lysate sample (20 uL) and add clarified lysate to equilibrated beads
6. Incubate with rotation for 1h at 4C
7. Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
8. Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS
9. Prepare elution buffer containing 10mM maltose in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.
10. Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay
11. Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
12. Measure protein concentration (Bradford Assay or Quantification by Absorbance at 280nm; concentrate if necessary and store at -20)
13. Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE