5. On the next day, harvest the cells by centrifugation at 8000 Â g,
4
C for 10 min. Pool the bacterial pellets of 3 Â 350 ml
cultures into one tube (see Note 4).
6. Resuspend the bacterial pellet of 1 l culture in 20 ml lysis buffer
by sonication (Duty Cycle 30/Output Control 2). 1 l bacteria
culture corresponds to approximately 2 g of cells after
pelleting.
7. After centrifugation (8000 Â g, 4
C, 10 min), resuspend the
pellet again in 20 ml lysis buffer by sonication. For each gram of
E. coli, add 2.5 μl Benzonase (10 U μl
À1 ) and 2.5 μl lysozyme
(10 mg ml
À1 ). Then incubate the cultures at 4
C for 30 min
on a shaker at 100 rpm.
8. Disintegrate by French press treatment (20,000 psi) and pellet
inclusion bodies by centrifugation at 16,000 Â g for 40 min at
4
C. Discard the supernatant.
9. Wash pellet containing inclusion bodies (IB) two times with
10 ml Millipore water, one time each with 1% Triton X-100 and
2 M urea. Resuspend pellets by sonication and recover by
centrifugation at 20,000 Â g for 10 min. at 4
C between
each step (see Note 5).
10. Resuspend pelleted inclusion body in 2 ml water by sonication
and add 10 ml IB-lysis buffer. Incubate for 30 min at room
temperature.
11. Slowly drop the resuspended inclusion bodies into 450 ml
refolding (RF) buffer cooled at 4
C, approximately one drop
every 3 s, while constantly stirring the buffer on ice. The
dripping can take up to 20 min. Continue stirring for another
20 min after the inclusion bodies have dripped into the RF
buffer (see Note 6).
12. Transfer RF solution containing 6ÂHis-PSY into a 500 ml
centrifugation bottle and pellet undissolved aggregates at
10,000 Â g for 20 min at 4
C. Carefully transfer supernatant
into empty bottle and discard the pellet.
13. Equilibrate 2 ml CoCl 2 Fractogel two times with RF buffer, as
described above for TALON equilibration for GGPS purification. Add equilibrated Fractogel to RF supernatant, place on
ice and incubate on a shaking platform for 20 min (see Note 7).
14. Centrifuge the solution at 700 Â g for 10 min and 4
C to
pellet the beads, carefully discard the supernatant by pipetting
and resuspend the beads in 10 ml PSY washing buffer. Transfer
the beads to a 15 ml plastic tube, centrifuge for 15 min at
700 Â g (4
C) and discard the supernatant by pipetting.
15. Resuspend the pellet in 3 ml PSY elution buffer, incubate for
20 min at 4
C in an overhead shaker and centrifuge at 700 Â g
for 5 min at 4
C.
Enzyme Activity of Phytoene Synthase
47
4
C for 10 min. Pool the bacterial pellets of 3 Â 350 ml
cultures into one tube (see Note 4).
6. Resuspend the bacterial pellet of 1 l culture in 20 ml lysis buffer
by sonication (Duty Cycle 30/Output Control 2). 1 l bacteria
culture corresponds to approximately 2 g of cells after
pelleting.
7. After centrifugation (8000 Â g, 4
C, 10 min), resuspend the
pellet again in 20 ml lysis buffer by sonication. For each gram of
E. coli, add 2.5 μl Benzonase (10 U μl
À1 ) and 2.5 μl lysozyme
(10 mg ml
À1 ). Then incubate the cultures at 4
C for 30 min
on a shaker at 100 rpm.
8. Disintegrate by French press treatment (20,000 psi) and pellet
inclusion bodies by centrifugation at 16,000 Â g for 40 min at
4
C. Discard the supernatant.
9. Wash pellet containing inclusion bodies (IB) two times with
10 ml Millipore water, one time each with 1% Triton X-100 and
2 M urea. Resuspend pellets by sonication and recover by
centrifugation at 20,000 Â g for 10 min. at 4
C between
each step (see Note 5).
10. Resuspend pelleted inclusion body in 2 ml water by sonication
and add 10 ml IB-lysis buffer. Incubate for 30 min at room
temperature.
11. Slowly drop the resuspended inclusion bodies into 450 ml
refolding (RF) buffer cooled at 4
C, approximately one drop
every 3 s, while constantly stirring the buffer on ice. The
dripping can take up to 20 min. Continue stirring for another
20 min after the inclusion bodies have dripped into the RF
buffer (see Note 6).
12. Transfer RF solution containing 6ÂHis-PSY into a 500 ml
centrifugation bottle and pellet undissolved aggregates at
10,000 Â g for 20 min at 4
C. Carefully transfer supernatant
into empty bottle and discard the pellet.
13. Equilibrate 2 ml CoCl 2 Fractogel two times with RF buffer, as
described above for TALON equilibration for GGPS purification. Add equilibrated Fractogel to RF supernatant, place on
ice and incubate on a shaking platform for 20 min (see Note 7).
14. Centrifuge the solution at 700 Â g for 10 min and 4
C to
pellet the beads, carefully discard the supernatant by pipetting
and resuspend the beads in 10 ml PSY washing buffer. Transfer
the beads to a 15 ml plastic tube, centrifuge for 15 min at
700 Â g (4
C) and discard the supernatant by pipetting.
15. Resuspend the pellet in 3 ml PSY elution buffer, incubate for
20 min at 4
C in an overhead shaker and centrifuge at 700 Â g
for 5 min at 4
C.
Enzyme Activity of Phytoene Synthase
47