3 Methods
3.1 Expression
and Purification
of Arabidopsis GGPP
Synthase 11
The following protocol yields approximately 1 mg recombinant
GGPS which is sufficient for a large number of PSY activity assays.
1. Add about 50 ng of plasmid DNA but maximally 1 μl volume
and 10 μl BL21 competent E. coli cells and mix gently by gentle
snipping. Do not vortex.
2. Incubate on ice for 5 min, then for 30 s at 42
C, place back on
ice for 5 min, add 250 μl SOC medium and incubate at 37
C
for 1 h while shaking at 200 rpm.
3. Plate 50 μl of transformed bacteria on LB-agar plates containing appropriate selective antibiotic. Incubate plate over night at
37
C.
4. Pick a single colony to inoculate a 37
C overnight culture of
15 ml of LB medium with appropriate selective antibiotics.
5. The next day, dilute the overnight culture 1:500 in 50 ml LB
medium containing 150 μg ml
À1 ampicillin, and grow at 37
C
to an OD 600 of 0.5.
6. Add 1 mM IPTG, and incubate for 4 h, then harvest by
centrifugation (5000 Â g, 5 min) and discard the supernatant.
The pellet can be stored at À20
C for several months if the
extraction cannot be performed the same day.
7. Resuspend the pellet in 10 ml GGPS buffer A and disrupt via a
French Press at 20,000 psi. Centrifuge at 10,000 Â g in plastic
centrifugation tubes for 10 min to pellet the cell debris. Decant
the supernatant into a new tube and discard the pellet.
8. Pipette 600 μl TALON resin slurry into a tube, add 10 ml
GGPS buffer A, centrifuge at 700 Â g for 1 min and discard
supernatant. Repeat washing one more time with 10 ml GGPS
buffer A.
9. Add the equilibrated TALON resin to the lysed bacterial, place
the tube horizontal in ice and incubate for 30 min while
shaking.
10. Load the suspension onto an empty 5 ml TALON column and
let the extract drain by gravity flow, then wash the retained
resin with 20 ml GGPS buffer A.
11. Elute the protein with 5 ml GGPS buffer B into 5 tubes with
each 1 ml eluate. Number tubes ascending. Figure 2a shows
steps of GGPS purification as well as purified GGPS loaded on a
Coomassie-stained SDS-PAGE gel.
Enzyme Activity of Phytoene Synthase
45
3.1 Expression
and Purification
of Arabidopsis GGPP
Synthase 11
The following protocol yields approximately 1 mg recombinant
GGPS which is sufficient for a large number of PSY activity assays.
1. Add about 50 ng of plasmid DNA but maximally 1 μl volume
and 10 μl BL21 competent E. coli cells and mix gently by gentle
snipping. Do not vortex.
2. Incubate on ice for 5 min, then for 30 s at 42
C, place back on
ice for 5 min, add 250 μl SOC medium and incubate at 37
C
for 1 h while shaking at 200 rpm.
3. Plate 50 μl of transformed bacteria on LB-agar plates containing appropriate selective antibiotic. Incubate plate over night at
37
C.
4. Pick a single colony to inoculate a 37
C overnight culture of
15 ml of LB medium with appropriate selective antibiotics.
5. The next day, dilute the overnight culture 1:500 in 50 ml LB
medium containing 150 μg ml
À1 ampicillin, and grow at 37
C
to an OD 600 of 0.5.
6. Add 1 mM IPTG, and incubate for 4 h, then harvest by
centrifugation (5000 Â g, 5 min) and discard the supernatant.
The pellet can be stored at À20
C for several months if the
extraction cannot be performed the same day.
7. Resuspend the pellet in 10 ml GGPS buffer A and disrupt via a
French Press at 20,000 psi. Centrifuge at 10,000 Â g in plastic
centrifugation tubes for 10 min to pellet the cell debris. Decant
the supernatant into a new tube and discard the pellet.
8. Pipette 600 μl TALON resin slurry into a tube, add 10 ml
GGPS buffer A, centrifuge at 700 Â g for 1 min and discard
supernatant. Repeat washing one more time with 10 ml GGPS
buffer A.
9. Add the equilibrated TALON resin to the lysed bacterial, place
the tube horizontal in ice and incubate for 30 min while
shaking.
10. Load the suspension onto an empty 5 ml TALON column and
let the extract drain by gravity flow, then wash the retained
resin with 20 ml GGPS buffer A.
11. Elute the protein with 5 ml GGPS buffer B into 5 tubes with
each 1 ml eluate. Number tubes ascending. Figure 2a shows
steps of GGPS purification as well as purified GGPS loaded on a
Coomassie-stained SDS-PAGE gel.
Enzyme Activity of Phytoene Synthase
45