10. Add 5 resin volumes of ATP Wash Buffer, Buffer D at a ratio of
3 mL per column, carefully mix, and then gently rotate at 4
C
for 30 min. Cap at the top and incubate at 4
C while rotating
the sample gently end over end (see Note 7). Let the resin settle
and then remove the bottom cap to allow most of the resin to
flow through, without letting the resin go dry. Cap the tube at
the bottom. Save this flow-through fraction to determine the
total protein content of unbound protein.
11. Elute MBP::Z-ISO from the resin using Elution Buffer, Buffer
E at a ratio of ~1 mL per liter of initial culture. Add 0.5 mL at a
time. Mix with entire resin and then elute into the collection
tube. Repeat eluting with 0.5 mL Buffer E until the resin is
clear (see Fig. 2c). The eluate should appear red (see Fig. 2d)
because the Z-ISO in MBP::Z-ISO carries a heme. After protein and heme quantification below, the best fractions can be
pooled (see Note 8).
12. Quantify the protein by absorbance (280–290 nm) and the
heme by absorbance (414–415 nm for oxidized ferric heme or
426 nm for reduced ferrous heme) using a NanoDrop instrument or a spectrophotometer. Blank the instruments with
Elution Buffer, Buffer E. For the NanoDrop, use settings of
ε ¼ 130.75 L mol
À1 cm
À1 and MW ¼ 75.46 kD. The expected
yield is ~5–6 mg L
À1 culture with ferric heme (414 nm) to
protein (280–290 nm) ratio of ~0.7–0.8 (which is about
1 heme to protein).
13. Optional: The protein can be further dialyzed depending upon
further analyses. Using new gloves, prewet a dialysis cassette
with Dialysis Buffer, Buffer F, kept at 4
C. Load the cassette
with the protein sample which has been concentrated, if necessary (see Note 9). Dangle the cassette in 2 L of precooled
Dialysis buffer, Buffer F, stirring overnight at 4
C. A
20 gauge needle can be used to remove the sample, taking
care not to damage the membrane. After dialysis, measure the
protein and heme concentration as described above using
Buffer F to blank the NanoDrop or spectrophotometer. The
sample can be further concentrated, depending upon the
downstream analysis (see Note 10).
4 Notes
1. Do not exceed 500 mL culture per 3 L Fernbach flask because
yields will be reduced.
2. Addition of heme biosynthesis precursors will improve heme
biosynthesis and double the heme loading of the MBP::Z-ISO
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
59
3 mL per column, carefully mix, and then gently rotate at 4
C
for 30 min. Cap at the top and incubate at 4
C while rotating
the sample gently end over end (see Note 7). Let the resin settle
and then remove the bottom cap to allow most of the resin to
flow through, without letting the resin go dry. Cap the tube at
the bottom. Save this flow-through fraction to determine the
total protein content of unbound protein.
11. Elute MBP::Z-ISO from the resin using Elution Buffer, Buffer
E at a ratio of ~1 mL per liter of initial culture. Add 0.5 mL at a
time. Mix with entire resin and then elute into the collection
tube. Repeat eluting with 0.5 mL Buffer E until the resin is
clear (see Fig. 2c). The eluate should appear red (see Fig. 2d)
because the Z-ISO in MBP::Z-ISO carries a heme. After protein and heme quantification below, the best fractions can be
pooled (see Note 8).
12. Quantify the protein by absorbance (280–290 nm) and the
heme by absorbance (414–415 nm for oxidized ferric heme or
426 nm for reduced ferrous heme) using a NanoDrop instrument or a spectrophotometer. Blank the instruments with
Elution Buffer, Buffer E. For the NanoDrop, use settings of
ε ¼ 130.75 L mol
À1 cm
À1 and MW ¼ 75.46 kD. The expected
yield is ~5–6 mg L
À1 culture with ferric heme (414 nm) to
protein (280–290 nm) ratio of ~0.7–0.8 (which is about
1 heme to protein).
13. Optional: The protein can be further dialyzed depending upon
further analyses. Using new gloves, prewet a dialysis cassette
with Dialysis Buffer, Buffer F, kept at 4
C. Load the cassette
with the protein sample which has been concentrated, if necessary (see Note 9). Dangle the cassette in 2 L of precooled
Dialysis buffer, Buffer F, stirring overnight at 4
C. A
20 gauge needle can be used to remove the sample, taking
care not to damage the membrane. After dialysis, measure the
protein and heme concentration as described above using
Buffer F to blank the NanoDrop or spectrophotometer. The
sample can be further concentrated, depending upon the
downstream analysis (see Note 10).
4 Notes
1. Do not exceed 500 mL culture per 3 L Fernbach flask because
yields will be reduced.
2. Addition of heme biosynthesis precursors will improve heme
biosynthesis and double the heme loading of the MBP::Z-ISO
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
59