6. Lennox Broth (LB) agar culture plates: 1% (w/v) Bacto tryptone, 0.5% (w/v) Bacto yeast extract, 0.5% (w/v) NaCl, 1.5 g
per 100 mL agar, deionized water, and 100 μg mL
À1 of ampicillin (see Note 3).
2.2 Heterologous
Expression of CCDs
in E. coli
1. 100 mg mL
À1 stock solution of ampicillin.
2. Lennox Broth (LB) media: 1% (w/v) Bacto tryptone, 0.5%
(w/v) Bacto yeast extract, 0.5% (w/v) NaCl, deionized water,
100 ug mL
À1 of ampicillin, and 2 M NaOH for pH 7.2
adjustment.
3. Baffled culture flasks (Pyrex) or Erlenmeyer flasks (see Note 4).
4. An agar plate with colonies of BL21 E. coli cells transformed
with the appropriate CCD expression plasmid.
5. Ferrous iron/ascorbic acid solution: 60 mg FeSO 4 and
10 mg L-ascorbate in 10 mL H 2 O (see Note 5).
6. 1 M stock solution of isopropyl β-D-thiogalactopyranoside
(IPTG).
7. Console incubator shaker (New Brunswick E25/E25R or
equivalent).
8. Cell density meter (Biochrom WPA CO8000 Cell Density Meter
or equivalent).
9. Cuvettes.
10. Centrifuge tubes/bottles (see Note 6).
11. Centrifuge (Eppendorf 5804 R or comparable model).
2.3 Preparation
of CCD Protein
Extracts
1. BL21 competent E. coli pellet.
2. Extraction buffer: 20 mM Tricine/KOH (pH 7.4) and
150 mM NaCl (see Note 7).
3. 10 mg mL
À1 stock solution of lysozyme.
4. 100 mM stock solution of Phenylmethylsulfonyl fluoride
(PMSF).
5. 100 mM stock solution of Tris (2-carboxyethyl) phosphine
hydrochloride (TCEP), pH 7.5.
6. 2 mL Eppendorf tubes.
7. Ultrasonic liquid processor (e.g., Q125 Sonicator from QSonica) (see Note 8).
8. Refrigerated centrifuge.
2.4 Small Scale
Purification
of Recombinant
6ÂHis-Tag-CCDs
1. Talon
® superflow metal affinity resin.
2. Binding buffer: 50 mM sodium phosphate, 300 mM NaCl,
1 mM TCEP, pH 7.0.
3. Wash buffer: 50 mM sodium phosphate, 300 mM NaCl,
10–20 mM imidazole, 1 mM TCEP, pH 7.0.
78
Linda Dora Thomas et al.
per 100 mL agar, deionized water, and 100 μg mL
À1 of ampicillin (see Note 3).
2.2 Heterologous
Expression of CCDs
in E. coli
1. 100 mg mL
À1 stock solution of ampicillin.
2. Lennox Broth (LB) media: 1% (w/v) Bacto tryptone, 0.5%
(w/v) Bacto yeast extract, 0.5% (w/v) NaCl, deionized water,
100 ug mL
À1 of ampicillin, and 2 M NaOH for pH 7.2
adjustment.
3. Baffled culture flasks (Pyrex) or Erlenmeyer flasks (see Note 4).
4. An agar plate with colonies of BL21 E. coli cells transformed
with the appropriate CCD expression plasmid.
5. Ferrous iron/ascorbic acid solution: 60 mg FeSO 4 and
10 mg L-ascorbate in 10 mL H 2 O (see Note 5).
6. 1 M stock solution of isopropyl β-D-thiogalactopyranoside
(IPTG).
7. Console incubator shaker (New Brunswick E25/E25R or
equivalent).
8. Cell density meter (Biochrom WPA CO8000 Cell Density Meter
or equivalent).
9. Cuvettes.
10. Centrifuge tubes/bottles (see Note 6).
11. Centrifuge (Eppendorf 5804 R or comparable model).
2.3 Preparation
of CCD Protein
Extracts
1. BL21 competent E. coli pellet.
2. Extraction buffer: 20 mM Tricine/KOH (pH 7.4) and
150 mM NaCl (see Note 7).
3. 10 mg mL
À1 stock solution of lysozyme.
4. 100 mM stock solution of Phenylmethylsulfonyl fluoride
(PMSF).
5. 100 mM stock solution of Tris (2-carboxyethyl) phosphine
hydrochloride (TCEP), pH 7.5.
6. 2 mL Eppendorf tubes.
7. Ultrasonic liquid processor (e.g., Q125 Sonicator from QSonica) (see Note 8).
8. Refrigerated centrifuge.
2.4 Small Scale
Purification
of Recombinant
6ÂHis-Tag-CCDs
1. Talon
® superflow metal affinity resin.
2. Binding buffer: 50 mM sodium phosphate, 300 mM NaCl,
1 mM TCEP, pH 7.0.
3. Wash buffer: 50 mM sodium phosphate, 300 mM NaCl,
10–20 mM imidazole, 1 mM TCEP, pH 7.0.
78
Linda Dora Thomas et al.