Purification of Amycolatopsis NSAR/OSBS
Last Updated by Andy McMillan 1-15-2015
A. Expression:
WT: 2 L LB + Carbenicillin + Kanamycin, 30 °C, 48 hours
Recommended: save an aliquot (equivalent to 5ml culture) for miniprep and
sequencing to verify the identity of the protein (good idea if you’re looking at
lots of mutants)
B. Weigh cell pellets in bottles before resuspension (and the bottles after removing
resuspended pellets).
C. Lysis
*** resuspension volume depends on lysis method:
Buffer A (binding buffer)
20 mM Tris, pH 8
5 mM MgCl2
For microfluidizer:
Resuspend in 20 ml Buffer A, 50 ul PMSF
add 300 ul DNase
For sonication:
Resuspend Cells in 40 ml Binding buffer A
add 80 l PMSF and 400 l DNase (~50X stock). Incubate on ice for 1 hour.
Sonicate Cells
50% Amplitude, 1s on/1s off pulse, 20 minutes. Larger pellets may need to
be split and/or require additional time after replacing ice
Resuspend in smaller volume without lysozyme for bead beater
C. Pellet the lysate – Oak ridge round bottom tubes (~45 ml), SS-34 rotor, 15,000
RPM, 30’, 4o
D. Filter Supernatant with 0.2 m filter (50 ml steriflip is easiest)
E. Set up FPLC (while cells are sonicating and spinning)
1. Make sure there is enough filtered ddH2O, 20% EtOH, and buffers A & B
buffer A (loading/binding buffer):
20 mM Tris, pH 8
5 mM MgCl2
buffer B (elution buffer):
20 mM Tris, pH 8
5 mM MgCl2
500 mM NaCl
2. Remove air from inlet tubing
3. PumpWash A & B w/ ddH2O
4. Also wash out tubing w/ 1-2 ml ddH2O (in load and inject positions)
5. Wash Column w/ ddH2O to remove ethanol (5 column volumes (CV); in load
position), then with buffers A and B
Column 1: HiPrep 16/10 DEAE FF (1 CV = 20 ml)
ddH2O - 5 CV, 5 ml/min
(pumpwash A &B with binding buffer (A) and elution buffer (B))
buffer A - 5 CV, 5 ml/min
buffer B (elution buffer) - 5 CV, 5 ml/min
(equilibrate with buffer A - 5 CV, 5ml/min- included in program)
6. Superloop should be already attached. See superloop protocol for cleaning and
loading sample details.
7. Connect inlet of superloop to position 6; fill 60 ml syringe w/ filtered sample and
inject into superloop; Connect superloop to position 2 (Do this with FPLC set on
Load; water in superloop will go to waste when sample is injected
8. Run program: glasnerHiPrepDEAE2step0131211 (step to 30% buffer B, gradient
from 30% Buffer B to 65% buffer B over 10 column volumes, than immediately up
to 100% B for 2 column volumes). If using to purify proteins with significantly
different charge than WT Amycolatopsis, these parameters may need to be
adjusted to get good purification
Column 1: HiPrep 16/10 DEAE FF
1 CV = 20 ml
maximum flow rate = 10 ml/min - 5 ml/min is recommended
max pressure = 0.15 Mpa + column backpressure (0.12 MPa) = 0.27 MPa
Equilibrate - 5 CV buffer A, 4 ml/min
Bind 1 ml/min
Wash out unbound - 2 CV, 2 ml/min
STEP - 30% B - 6 CV, 5 ml/min - collect in 50 ml tubes
Gradient - 30%-65% B, 10 CV, 2.5 ml/min -- collect in 10 ml tubes
Wash tight binding (should be very little) – 100% B for 2 column
volumes
9. Collect fractions containing target protein. Run samples on gel (see J). Select
samples from the region where the protein is expected to come off (usually start
around 9th fraction, and load every fraction.
F. Clean DEAE Column and prepare FPLC for next column.
***REMEMBER TO SET GRADIENT TO 100% B FOR EACH STEP
1. Switch B pump intake to 2 M NaCl and Pumpwash B.
2. wash DEAE column with 40 ml 2 M NaCL @ 5 ml/min to remove remaining
protein.
3. Switch B pump intake to water and Pumpwash B. Wash column with water until
UV and conductivtiy baselines are stable (at least 5 CV).
4. Switch B pump intake to 20% ethanol and Pumpwash B. Wash column with 5 CV
20% ethanol for storage.
5. If possible, start the second column to run overnight. Otherwise, the system
can be left like this until morning. DO NOT leave the FPLC in running
buffers longer than overnight.
G. Running the phenylsepharose column. Run at 4 oC
1. Switch B pump intake to water and pumpwash B.
2. Prepare 2nd column
Use 3 linked 5 ml HiTrap Phenyl FF (low sub) columns
Wash with water - 3 x 5 CV = 15 CV, 5 ml/min
Wash with Buffer A (3 x 5 CV = 15 CV) (low salt buffer), 5 ml/min
3. Switch B pump intake to Buffer C (high salt) and pumpwash B.
Buffer C
20 mM Tris, pH 8
5 mM MgCl2
0.4 M (NH4)2SO4
4. Pool fractions from DEAE columns and bring to 0.4 M (NH4)2SO4 using a 3 M
(NH4)2SO4 stock. Add it slowly and mix the solution as you go, so that the protein
doesn’t precipitate. Filter the sample and load onto superloop.
1 fraction = 1.53 mL
2 fractions = 3.08 mL
3 fractions = 4.61 mL
4 fractions = 6.15 mL
5. Run Program: glasnerPhenylLow3col062011
Equilibrate - 3 x 5 CV = 15 CV Buffer C (high Salt)
Bind - 0.25 ml / min
Wash (Buffer C (high Salt)) - 3 x 2 CV = 6 CV @ 0.25 ml/min
Elute - 1 ml/min,
step 1: 100% B (buffer C, high salt), 3 x 2 CV = 6 CV
Step 2: 100%-0% B, 3 x 10 CV = 30 CV
6. Collect Fractions, run on gel
*** One mutant we tried to purify was expressed at very low levels and copurified
with a larger band. We may need to follow this column with size exclusion
chromatography.
7. Clean/regenerate columns by running 3 x 5 CV = 15 CV 1 M NaOH, then 5 CV
water ate 1 ml/min. Put the NaOH and H2O on the B pump, because you will be
using Buffer A again in the final step
8. If you won’t be running another column immediately, you need to switch the whole
system to water, then ethanol, as usual.
H. Check activity of fractions
I’ve noticed some variability in fractions, even if bands appear similar. To ensure we
collect active protein check the specific activity of fractions.
1. Template Protocol is included in both PhenylsepharoseFractions and
CombinedKinetics protocols on the plate reader computer (the later also has
template for standard Michaelis-Menton characterization)
2. Measure protein concentration with 10 uL from fractions and 90 uL of GuHCl
3. Setup reaction buffer in loading channels (100 uL of 1mM MnCl2, 100 uL Tris pH
8.0, 200 uL of SHCHC (generally use 7500 uM, but other can work if near
saturating), water to 1000 uL - (10 x volume from fraction using) total volume
4. Load fractions into plate, 1uL works for active protein, some less active mutants
require more to have a noticeable change
Buffer exchange and concentration
1. Pool fractions with pure protein
2. Exchange buffer by repeatedly concentrating sample and resuspend with buffer A.
After (NH4)2SO4 has been sufficiently diluted, concentrate to 0.5 – 1.0 mL
4. Measure OD280 to get protein concentration.
5. Add glycerol to a final concentration of ~25-30%.
6. Store at -20 C.
7. calculate protein yield (mg protein/L of culture)
I. Clean up FPLC
1. Move pump intakes to 20% EtOH; do PumpWash (should be in water from earlier
steps)
3. Wash column w/ 20% EtOH for 5 VC (25 ml) @ 3 ml/min (or at rate designated for
column); Remove column and cap and parafilm ends; label w/ protein and date
and store @ 4o
4. Wash superloop as described in other procedure
5. do final wash  PumpWash; Load Wash and Inject Wash (2-5 ml each)
6. Empty waste containers
J. Run samples on gel (10% Bis Tris)
Lysate and pellet:
0.5 l sample
14.5 l binding buffer A or water
5 l 4X Loading dye
2 l 1 M DTT
Other Fractions:
15 l sample
5 l 2X Loading dye
2 l 1 M DTT
Heat at 70º for 10’
Run gel 200V, 50’
Use Denville Blue or fast stain/destain procedure so you can pool fractions
ASAP. For active protein variants, you can also assay fractions to select them.