Purifying Protein from Inclusion Bodies
|50 mM tris-HCl, pH 8.0
||20 mM Na2HPO4, pH 7.2
|5 mM EDTA
||20 mM NaCl
|10 mM NaCl
||5 mM EDTA
||25% (w/v) sucrose
- Weigh the centrifuge bottle (w/cap) before adding
culture media. You will need to know how many grams
of cells you have before you start lysis.
- Spin down cells from large culture at 6000 rpm for
20 min. If you have a lot of media you can do multiple
spins pouring out the supernatant after each spin.
- When spinning is complete weigh the flask (w/cap)
and the cell pellet. Subtract the total weight from
the empty flask weight to determine how many grams
of cells you have.
- Add 3 mL of buffer A to the flask for each gram
of cells and fully resuspend the cell pellet.
- Transfer the suspended pellet to 50 mL conical centrifuge
- Add enough 100 mM PMSF to make a 1 mM final solution
(10ul 100 mM PMSF/ml of solution).
- Add 16 ul of 50mg/ml lysozyme per gram of cells
- Place in 37oC water bath until solution becomes
- Sonicate the solution to chop up the DNA and reduce
- Spin solution at 18,000 rpm for 30 min. Save supernatant.
- Add 3 mL of buffer B to the tube for each gram of
cells and fully resuspend the cell pellet.
- Add the same amount of 100 mM PMSF as you did in
step 6 and 10 ul Triton X-100 per ml of solution and resuspend the pellet.
- Spin at the highest speed possible (~20,000 rpm)
for 20 min. Save supernatant. The pellet remaining
will be the inclusion bodies.
- Add 20 mL of 8 mM urea (add DTT to 8 mM urea if
purifying proteins with cysteine residues) and dissolve
the pellet. Heating in a bath (37-50oC) may facilitate
the pellet dissolving.
- Repeat step 14 until no more of the pellet dissolves.
- Dialyze 8 M urea solutions in 4 L of a 50 mM tris-HCl
buffer solution at pH 8.5 using 3.5 kD molecular weight
cutoff dialysis tubing for 2 days. Repeat, dumping
out buffer and replacing with fresh buffer.
- Empty dialysis tubing into 50 mL conical centrifuge
tubes and spin at 20,000 rpm for 30 min. Freeze supernant until HPLC purification.
- Clean up and wash all centrifuge tubes and flasks
that you used.
Next step is to HPLC purify the protein. Before
this step the solutions MUST be filtered through a
0.2u or 0.45u filter.