1. Prewarm the medium to 37
C. For 4 Â 500 mL each in 3 L
Fernbach, add 5 mL overnight culture per 500 mL of 2Â YT
medium plus antibiotic, 0.5 mL ampicillin stock/500 mL.
Incubate for 1 h without shaking and then shake at
220 rpm, until OD 600 equals 0.6 (~3 h) (see Note 1).
2. IPTG induction with heme biosynthesis reagents (see Note 2):
For each 500 mL, add 0.5 mL each of 1 M IPTG, 235 mM
ALA, and 35 mM ferrous iron. Incubate for ~16 h at 28
C
with shaking.
DAY 3
1. Centrifuge cells at 2600 Â g (3,500 rpm, Eppendorf centrifuge
5810 R) for 15 min at 4
C and freeze (À80
C) pellets until
use. Use only 2 centrifuge bottles and spin twice to combine
cultures (see Note 3).
DAY 4
1. Resuspend in ~80 mL Buffer B (made with Buffer A and freshly
added components (see Note 4).
2. Sonicate the entire sample on ice in a 250 mL plastic beaker (10
times, 30 s each, 60% duty cycle with pulsing (set at “8”), with
1 min between sonication) using a Vibra Cell VC600 sonicator
equipped with a FLAT tip (Sonics & Materials Inc., Connecticut, USA). The sample should change from milky white to a
translucent red (see Fig. 2a and Note 5).
3. Centrifuge at ~9800 Â g (11,000 rpm, in a Type 60 Ti rotor)
for 15 min, 4
C (Polycarbonate tubes, 25 Â 89 mm, Beckman
Coulter). Remove supernatant with pipet and save lysates containing the membrane fraction. The pellet will contain unbroken cells.
4. Centrifuge lysates at ~83,000 Â g (32,000 rpm, in a Type 60 Ti
rotor) for 1 h at 4
C using Polycarbonate tubes (25 Â 89 mm,
Beckman Coulter). Invert tubes to drain remaining supernatant. The pellets should appear bright red (see Fig. 2b and Note
6).
5. Resuspend pellets in 16 mL Resuspension Buffer C t ratio of
8 mL per liter of initial cell culture. Use a glass homogenizer.
Do not over homogenize.
6. Increase the volume to a total of 80 mL final at a ratio of 40 mL
of Buffer C per liter of starting culture. Divide the sample into
two 50 mL Falcon tubes and add to each 0.6 g DDM as a
powder (1.5% final concentration). To dissolve the DDM into
the buffer, slowly rotate the samples end over end at 4
C for
15 min using a rotator machine.
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
57
C. For 4 Â 500 mL each in 3 L
Fernbach, add 5 mL overnight culture per 500 mL of 2Â YT
medium plus antibiotic, 0.5 mL ampicillin stock/500 mL.
Incubate for 1 h without shaking and then shake at
220 rpm, until OD 600 equals 0.6 (~3 h) (see Note 1).
2. IPTG induction with heme biosynthesis reagents (see Note 2):
For each 500 mL, add 0.5 mL each of 1 M IPTG, 235 mM
ALA, and 35 mM ferrous iron. Incubate for ~16 h at 28
C
with shaking.
DAY 3
1. Centrifuge cells at 2600 Â g (3,500 rpm, Eppendorf centrifuge
5810 R) for 15 min at 4
C and freeze (À80
C) pellets until
use. Use only 2 centrifuge bottles and spin twice to combine
cultures (see Note 3).
DAY 4
1. Resuspend in ~80 mL Buffer B (made with Buffer A and freshly
added components (see Note 4).
2. Sonicate the entire sample on ice in a 250 mL plastic beaker (10
times, 30 s each, 60% duty cycle with pulsing (set at “8”), with
1 min between sonication) using a Vibra Cell VC600 sonicator
equipped with a FLAT tip (Sonics & Materials Inc., Connecticut, USA). The sample should change from milky white to a
translucent red (see Fig. 2a and Note 5).
3. Centrifuge at ~9800 Â g (11,000 rpm, in a Type 60 Ti rotor)
for 15 min, 4
C (Polycarbonate tubes, 25 Â 89 mm, Beckman
Coulter). Remove supernatant with pipet and save lysates containing the membrane fraction. The pellet will contain unbroken cells.
4. Centrifuge lysates at ~83,000 Â g (32,000 rpm, in a Type 60 Ti
rotor) for 1 h at 4
C using Polycarbonate tubes (25 Â 89 mm,
Beckman Coulter). Invert tubes to drain remaining supernatant. The pellets should appear bright red (see Fig. 2b and Note
6).
5. Resuspend pellets in 16 mL Resuspension Buffer C t ratio of
8 mL per liter of initial cell culture. Use a glass homogenizer.
Do not over homogenize.
6. Increase the volume to a total of 80 mL final at a ratio of 40 mL
of Buffer C per liter of starting culture. Divide the sample into
two 50 mL Falcon tubes and add to each 0.6 g DDM as a
powder (1.5% final concentration). To dissolve the DDM into
the buffer, slowly rotate the samples end over end at 4
C for
15 min using a rotator machine.
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
57