Preparation of Phosphate Binding Protein (PBP) from E. coli (modified cell line
from Ed Taylor)
Day #1
1. 200 ml of LB media was autoclaved and cooled to 37 oC. 0.2 ml of 12.5 mg/ml filter
sterilized tetracycline was added to the media to adjust to 12.5 mg/liter tetracycline.
2. 10 ml of the media were placed in a 100 ml flask while 90 ml were placed in a sterile
500 ml flask.
3. A vial of glycerol stock E. coli (frozen at -80 oC) was thawed in a 37 oC water bath.
The contents of the stock vial were inoculated into the 100 ml flask containing 10 ml of
LB with tetracycline. The flask was rapidly shaken (120 rpm) in an upright position with
the cap vented at 37 oC.
Growing of Cells for Preparation of PBP
4. After 6 hrs of incubation, the contents of the 100 ml flask were transferred to the flask
containing 90 ml of LB media + tetracycline. This was further incubated with rapid
shaking for 16 hrs (overnight).
5. Prepare four liters of Minimal Media as follows:
100 mM HEPES*
20 mM KCl
15 mM (NH4)2SO4
1 mM MgCl2
10 M FeSO4
1 g/ml thiamine
0.25 % glycerol (v/v)
2 mM KH2PO4
95.32 g
5.968 g
7.926 g
0.810 g
11.12 mg
4.0 mg
10 ml
1.089 g
The above media was prepared and the pH was adjusted to 7.5. 500 ml aliquots were
placed in each of 8 x 2 liters flasks and the media was autoclaved. Once cooled, 0.6 ml
of 12.5 mg/ml tetracycline (dissolved in 50 % ethanol) was added to each flask to adjust
15 mg/l. The media was then ready for use. * The procedure called for TES, however, the yields
have been reported to be better when HEPES was used. HEPES has a similar pKa and is most effective
over the same pH buffering range so it was used instead.
Day #2
1. Following overnight incubation, the 100 ml of E. coli culture was diluted by placing 10
ml into each of the 8 two liter baffled flasks containing 500 ml of Minimal Media
equilibrated to 37 oC. The flasks were incubated at 37 oC with rapid shaking.
2. The optical density of the culture at 600 nm was closely followed.
Time, hr
OD600 vs. media
net change of OD
0
0.077
0
1
0.110
0.0328
2
0.2175
0.1403
At 2hrs of incubation, 5 ml of filter sterilized 200 mM rhamnose ( adjusted to pH 7.5
before use) was added to each flask (final [rhamnose] = 2 mM). After the rhamnose
added, the culture were incubated overnight at 37 oC with rapid shaking.
Day #3
1. Following overnight incubation in Minimal media + rhamnose (final OD600 ~2.04), the
cells were collected from each 500 ml of culture by spinning at 4,000 rpm for 20 min at
room temp. in a SLA-3000 rotor. The pelleted cells were then subjected to osmotic
shock to release the phosphate binding protein.
Osmotic Disruption of E.coli Cells to Yield PBP:
2. The pellets were resuspended in a total volume of 650 ml of 10 mM TrisHCl (pH
7.6)/30 mM NaCl at room temp. The pellets were combined and spun again at 4,000 rpm
for 20 min at room temp.
3. The resulting supernatant was discarded. The pellets were resuspended in 350 ml of
the same buffer, transferred to two pre-weighed 250 ml centrifuge bottles and spun at
5,000 rpm for 20 min.
4. The supernatant was discarded (care should be taken when decanting the supernatant at
the pellet tends to be very loose at this point) and weigh the wet pellet (
g).
5. Following the washes, the cell pellets were resuspended in 100 ml of 33 mM TrisHCl
(pH 7.6) at room temp. and transferred to a single 250 ml centrifuge bottle containing a
stirring bar. 100 ml of 40 % sucrose/ 0.1 mM EDTA/33 mM TrisHCl (pH 7.6) was
added while stirring rapidly. This was left at 25 oC to stir for 10 min.
6. The suspension was then spun at 10,000 rpm (SLA 600 rotor) for 20 min at 4 oC. The
resulting supernatant was discarded.
7. The pellet was rapidly resuspended in 200 ml of ice cold 0.5 mM MgCl2. Once the
pellet was removed and saved for PAGE from the suspension by spinning at 10,000 rpm
for 20 min, the resulting supernatant was removed and saved and measured to be ___ ml
(It was noted that the supernatant had a distinct purple tint.). Determine the concentration
of the supernatant to obtain the yield (total yield ______ mg).
8. SDS-PAGE: Run the pre-cast gel (10 % Tris Gly gel) with the samples (PBP released
from the cells at step 7, along with a sample* of the E. coli pellet remaining after the
osmotic shock procedure, step 7, to determine its PAGE profile.
*To prepare the pellet sample for use on the gel, 0.1 g (wet weight) of pellet was resuspended in 0.5 ml of
10 mM TrisHCl (pH 7.6). This was adjusted to 1 % 2-mercaptoethanol with the addition of 5 l of 2-ME
and boiled for 10 min. The suspension was spun at 10,000 rpm for 10 min to remove the cells. The
concentration of the supernatant was determined by Bradford assay.
9. The PBP (supernatant obtained at step 7) was directly loaded onto Q sepharose after
adjusting to tris 10 mM pH 7.6. Further purification is with Q-sepharose.