Pfu expression and purification
(based on Amity Manning’s preparation, 2003)
Expression

Transform 6His-Pfu (pET166) into BL21 DE3 cells.

Inoculate several fresh colonies into 5ml LB Amp culture.

Inoculate 1L LB Amp w/5ml culture.

Grow to OD  1.0 at 37°C, shaking at 2,000 rpm.

IPTG induce (see related protocol) w/1.5mM IPTG for 12 hr. at 37°C.

Pellet cells (5K rpm, 4°C in GSA rotor).

Resuspend cells with PBS and re-pellet.

Resuspend in 10ml Buffer A [50mM Tris pH 7.4, 10% glycerol], transfer to
Oakridge tube and freeze at –80°C.
Purification

Thaw pellet and add 0.1mg/ml lysozyme (200ml of 10mg/ml stock).

Add 0.01% NP-40 (2µl).

Incubate at 4°C for 30 min. (on ice).

Add 5mM MgSO4 (100µl 1M MgSO4).

Add 0.1% NP-40 (20µl).

Add 10µg/ml DNAse I (2µl 10mg/ml stock).

Incubate at 4°C for 30min (on ice).

Sonicate 6 x 30sec on ice (large probe, 80 watts).

Spin in SS34 rotor to generate MSS#1 (12,000 rpm, 20 min., 4°C).

Remove supe (discard pellet) and take sample for gel.

Incubate supe at 85°C for 30 min.

Spin in SS34 rotor to generate MSS#2 (17,500 rpm, 20 min., 4°C).

Remove supe (discard pellet after gel is run) and take sample of supe for gel. Supe
volume should be ~15ml.

Run samples before and after 85°C incubation on 12% gel: Load 5, 3, 2, 1, 0.5µl
each supe onto gel. Most protein pellets after 85°C incubation; Pfu remains in the
supe! (see protein database entry for gel example).

Pre-equilibrate 3ml gravity flow Ni column with 20ml buffer B + 30mM
imidazole (recipe below).

Load supe onto Ni column. Collect and save the flow through.

Wash unbound protein with 30ml of buffer B + 60mM imidazole. Collect and
save the wash.

Elute Pfu with 10ml buffer B + 250mM imidazole, collecting 1.0ml fractions.
Run a gel (loading 2µl of each fraction) and Coomassie stain.

Pool peak Pfu-containing fractions (usually ~6ml) and dialyze against 1L HEK
buffer [20mM Hepes pH=7.5, 1mM EDTA, 50mM KCl] overnight in cold room.

Load sample onto S HiTrap 5ml column (AP Biotech) equilibrated in HEK
buffer.

Elute with 20ml gradient (50mM – 1M KCl) in HEK buffer and collect 3ml
fractions.

Run 2µl of flow through, wash, and each fraction on a 12% gel, Coomassie stain.

Collect peak fractions and concentrate to 1.5ml in Centricon-30 device.

Dialyze vs. buffer C [100mM Tris pH 8.2 / 0.1mM EDTA / 1mM DTT], 3
changes, > 3 hr. each.

Dilute 1.5ml to 3ml with 1.5ml of buffer D [glycerol (100%), 0.1mM EDTA,
1mM DTT, 0.2% Igepal, 0.2% Tween20].

Thus, the final storage buffer is: 50mM Tris pH 8.2, 0.1mM EDTA, 1mM DTT,
0.1% Igepal, 0.1% Tween20, 50% glycerol.

Aliquot in 30 x 100µl and store at –20°C until activity is confirmed (long-term
storage at –80°C).
Phosphate buffers (for Ni column)
2X Buffer B (200ml):
Imidazole additions:

1.2ml 1M Na2HPO4
Final

17.6ml 1M NaH2PO4
30mM:
25ml

24ml 5M NaCl
60mM:
25ml
1.5ml
23.5ml

157.2ml ddH2O
250mM:
25ml
6.25ml
18.75ml
2X buffer 2M imidaz.
750µl
ddH2O
24.25ml