summary, purification of the 75.46 kD MBP::Z-ISO expressed in
E. coli was accomplished with fivefold improvement of yield and
doubled the heme content as compared to the previously published
method [5]. Per liter culture, this newer method describes a yield of
purified MBP::Z-ISO of 5–6 mg protein with heme to Z-ISO ratio
of ~ 1:1.
2 Materials
1. 1,4-dithiothreitol (DTT).
2. 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
(AEBSF).
3. n-Dodecyl β-D-maltoside (DDM) Sol grade.
4. Adenosine triphosphate (ATP).
5. Lysozyme from chicken egg white.
6. Sodium chloride (NaCl).
7. Tris.
8. Yeast extract.
9. Tryptone.
10. Ni-NTA agarose.
11. Slide-A-Lyzer dialysis cassettes G2 20K.
12. Glycerol.
13. Bottle, with cap assembly, polycarbonate, 26.3 mL,
25 Â 89 mm, 1 Â 3–1/2.
14. Ampicillin (50 mg/mL), filter sterile.
15. Ethylenediaminetetraacetic acid (EDTA).
16. Magnesium chloride (MgCl 2 ).
17. 5-aminolevulinic acid hydrochloride (ALA)
18. Ammonium iron (II) sulfate hexahydrate (ferrous iron).
19. YT medium (2Â yeast extract and tryptone): 1% yeast extract,
1.6% tryptone, and 0.5% NaCl, sterilized for 20
0 .
20. 1 M isopropyl-beta-D-thiogalactopyranoside (IPTG): 2.38 g/
10 mL. Filter-sterilize. Aliquot 4 mL per tube. Freeze until use.
21. 5-Aminolevulinic Acid Hydrochloride (ALA): 1000Â Stock:
235 mM ALA (39.3 mg/mL). Filter-sterilize. Keep on ice
until used.
22. Ferrous iron. ammonium iron (II) sulfate hexahydrate: 1000Â
Stock 35 mM (13.7 mg/mL), weigh ~50 mg into 3.65 mL
water. Filter-sterilize. Keep on ice until used.
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
55
E. coli was accomplished with fivefold improvement of yield and
doubled the heme content as compared to the previously published
method [5]. Per liter culture, this newer method describes a yield of
purified MBP::Z-ISO of 5–6 mg protein with heme to Z-ISO ratio
of ~ 1:1.
2 Materials
1. 1,4-dithiothreitol (DTT).
2. 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride
(AEBSF).
3. n-Dodecyl β-D-maltoside (DDM) Sol grade.
4. Adenosine triphosphate (ATP).
5. Lysozyme from chicken egg white.
6. Sodium chloride (NaCl).
7. Tris.
8. Yeast extract.
9. Tryptone.
10. Ni-NTA agarose.
11. Slide-A-Lyzer dialysis cassettes G2 20K.
12. Glycerol.
13. Bottle, with cap assembly, polycarbonate, 26.3 mL,
25 Â 89 mm, 1 Â 3–1/2.
14. Ampicillin (50 mg/mL), filter sterile.
15. Ethylenediaminetetraacetic acid (EDTA).
16. Magnesium chloride (MgCl 2 ).
17. 5-aminolevulinic acid hydrochloride (ALA)
18. Ammonium iron (II) sulfate hexahydrate (ferrous iron).
19. YT medium (2Â yeast extract and tryptone): 1% yeast extract,
1.6% tryptone, and 0.5% NaCl, sterilized for 20
0 .
20. 1 M isopropyl-beta-D-thiogalactopyranoside (IPTG): 2.38 g/
10 mL. Filter-sterilize. Aliquot 4 mL per tube. Freeze until use.
21. 5-Aminolevulinic Acid Hydrochloride (ALA): 1000Â Stock:
235 mM ALA (39.3 mg/mL). Filter-sterilize. Keep on ice
until used.
22. Ferrous iron. ammonium iron (II) sulfate hexahydrate: 1000Â
Stock 35 mM (13.7 mg/mL), weigh ~50 mg into 3.65 mL
water. Filter-sterilize. Keep on ice until used.
Purification of the Carotenoid Biosynthetic Enzyme Z-ISO
55