5. The addition of ferrous iron to the culture medium significantly increases the yield of enzymatically active protein.
6. Depending on the expression volume, 50 mL centrifuge tubes
may be used for a 50 mL culture. For larger volumes, the
appropriate sized centrifuge bottles are required.
7. The volume of extraction buffer that is needed will depend
both on the volume of cultures and the bacterial density of
cultures. We routinely use 2 mL of extraction buffer for a
bacterial pellet harvested from a 50 mL of bacterial culture.
8. The diameter of the sonicator probe will determine the volume
of sample that can be lyzated. For example, the Q125 has a
3 mm diameter probe and thus, can sonicate 500 μL to 15 mL
of liquid. For larger volumes a sonicator with a larger probe is
required.
9. The amount of acetone can be varied depending on the polarity
of the carotenoid substrate. Carotenes need less acetone, while
xanthophylls need more to achieve proper separation.
10. Various carotenoid substrates can be purified using Thin Layer
Chromatography.
11. DMN needs to be prepared fresh each time. Other detergents
such as n-Octyl β-D-thioglucopyranoside can be used as well.
The amount of the detergents in the enzyme assay must exceed
the critical micellar concentration of the respective substance.
12. The separation of soluble from insoluble protein fractions is
mandatory. In our hands, only the soluble fraction (supernatant) will contain enzymatically active CCD. The insoluble
fraction may contain significant amounts of CCD protein.
However, this protein fraction does not display enzymatic
activity and likely exists in inclusion bodies. SDS polyacrylamide electrophoresis and staining of the gels with Coomassie
Brilliant Blue may give hints about the distribution of the
recombinant CCD when expressed in significant amounts.
Western blotting can further help to determine the distribution
of the CCD in soluble and insoluble fractions when antibodies
against the CCD and/or fusion parts (polyhistidine-tag etc.) of
the recombinant protein are available.
13. The amount of lysate depends on the expression levels and
solubility of the recombinant CCD. Lower amounts might be
used when the protein exist in high levels in the soluble fraction, whereas higher amounts are needed when the CCD is
expressed at low levels.
14. We routinely use up to 2000 pmol carotenoid substrate per
100 μL enzyme assay. This amount of substrate can be solubilized in DMN detergent micelles. The concentration of the
carotenoid solution can be determined spectrophotometrically
86
Linda Dora Thomas et al.
6. Depending on the expression volume, 50 mL centrifuge tubes
may be used for a 50 mL culture. For larger volumes, the
appropriate sized centrifuge bottles are required.
7. The volume of extraction buffer that is needed will depend
both on the volume of cultures and the bacterial density of
cultures. We routinely use 2 mL of extraction buffer for a
bacterial pellet harvested from a 50 mL of bacterial culture.
8. The diameter of the sonicator probe will determine the volume
of sample that can be lyzated. For example, the Q125 has a
3 mm diameter probe and thus, can sonicate 500 μL to 15 mL
of liquid. For larger volumes a sonicator with a larger probe is
required.
9. The amount of acetone can be varied depending on the polarity
of the carotenoid substrate. Carotenes need less acetone, while
xanthophylls need more to achieve proper separation.
10. Various carotenoid substrates can be purified using Thin Layer
Chromatography.
11. DMN needs to be prepared fresh each time. Other detergents
such as n-Octyl β-D-thioglucopyranoside can be used as well.
The amount of the detergents in the enzyme assay must exceed
the critical micellar concentration of the respective substance.
12. The separation of soluble from insoluble protein fractions is
mandatory. In our hands, only the soluble fraction (supernatant) will contain enzymatically active CCD. The insoluble
fraction may contain significant amounts of CCD protein.
However, this protein fraction does not display enzymatic
activity and likely exists in inclusion bodies. SDS polyacrylamide electrophoresis and staining of the gels with Coomassie
Brilliant Blue may give hints about the distribution of the
recombinant CCD when expressed in significant amounts.
Western blotting can further help to determine the distribution
of the CCD in soluble and insoluble fractions when antibodies
against the CCD and/or fusion parts (polyhistidine-tag etc.) of
the recombinant protein are available.
13. The amount of lysate depends on the expression levels and
solubility of the recombinant CCD. Lower amounts might be
used when the protein exist in high levels in the soluble fraction, whereas higher amounts are needed when the CCD is
expressed at low levels.
14. We routinely use up to 2000 pmol carotenoid substrate per
100 μL enzyme assay. This amount of substrate can be solubilized in DMN detergent micelles. The concentration of the
carotenoid solution can be determined spectrophotometrically
86
Linda Dora Thomas et al.