4. Elution buffer: 50 mM sodium phosphate, 300 mM NaCl,
250 mM imidazole, 1 mM TCEP, pH 7.0.
5. Amicon
® Ultra—0.5 mL 10 K centrifugal filters or similar.
2.5 Carotenoid
Substrate Preparation
for CCDs Enzymatic
Assay
1. Thin Layer Liquid Chromatography (TLC) developing tank.
2. TLC solution for carotenoid purification: 30 mL of acetone,
50 mL of Petroleum ether (PE), and 20 mL of Diethyl ether
(DEE) (see Note 9).
3. Thin Layer Chromatography plates (Millipore Sigma TLC Silica gel 60 F254).
4. Ruler and pencil.
5. 2 mL solvent tight reaction tubes.
6. Chloroform.
7. Carotenoids (see Note 10).
8. Glass cylinder tube.
9. Flat edge spatula.
10. Dark-colored glass storage bottle.
11. 10 Â 10 cm Aluminum foil.
12. UV spectrophotometer (Shimadzu UV-1280 or equivalent).
13. Concentrator (Vacufuge plus from Eppendorf or similar).
2.6 Enzymatic Assay
for Recombinant CCDs
1. Decyl maltose neopentyl glycol (DMN): 3% (w/v) of DMN in
100% ethanol (see Note 11).
2. Carotenoid substrate.
3. Eppendorf tubes.
4. Bacterial protein extract and/or purified CCD enzyme.
5. Diethyl ether (DEE).
6. Petroleum ether (PE).
7. Acetone.
8. Deionized water.
9. Nonrefrigerated microcentrifuge.
10. Thermomixer (Eppendorf).
11. Concentrator (Vacufuge plus from Eppendorf or similar).
3 Methods
3.1 CCD Expression
Plasmid
Transformation
into E. coli
1. Mix 1 μL of CCD expression plasmid (approx. 50 ng of DNA)
and 10 μL of BL21 competent E. coli cells (Stratagene)
together by gently stirring. Do not mix by pipetting.
2. Incubate on ice for 5 min.
Mammalian Carotenoid Cleavage Dioxygenases
79