104
3 Methods
- Transform BL21 (DE3) with the CED-3-expressing con-
struct, inoculate a single transformed colony in 10 ml of LB
medium containing 50 μg/ml ampicillin, and grow at 37 °C
with shaking at 200 rpm overnight. - Transfer 10 ml LB culture into 2 L of LB medium with 50 μg/
ml ampicillin and incubate at 37 °C with shaking at 200 rpm
until OD 600 of the culture reaches 0.5. - As soon as the OD 600 of the bacterial culture reaches 0.5, place
the culture on ice for 10 min. - Add IPTG to the bacterial culture to a fi nal concentration of
1 mM. - Incubate the culture at room temperature with shaking at
200 rpm for 4 h. - Place the bacterial culture on ice for 10 min and spin down the
cells at 4,500 × g for 10 min at 4 °C. - Discard the supernatant and move the pellet immediately to
−80 °C freezer for at least 1 h ( see Note 6 ). - Resuspend the frozen pellet in 10 ml of the cold sonication
buffer. This should be performed at 4 °C. - Perform sonication to break up the resuspended cells until
cleared lysate without viscosity is obtained ( see Note 7 ). It is
important to keep the lysate at low temperature. - Centrifuge the lysate at 18,000 × g , 4 °C, for 30 min and fi lter
the supernatant using a syringe and a 0.45 μm fi lter. - Place 0.3 ml bed volume of Ni-NTA resin in a plastic column
and wash the resin with 2 ml of the washing buffer. After this
step, all procedures should be performed at 4 °C. - Load the fi ltered lysate onto the column and wait until all
lysate has fl own out of the resin. - Wash the column with 20 ml of the washing buffer ( see Note 8 ).
- Elute CED-3::His 6 from the resin by adding 1 ml of elution
buffer 1. Repeat this procedure by adding 1 ml of elution buf-
fer 2 and 1 ml of elution buffer 3 each ( see Note 9 ). - Add 1 μl of purifi ed CED-3 into a total 19 μl of reaction
mixture, which contains 5 μM Ac-Asp-Glu-Val-Asp-AMC ( see
Note 10 ) and 10 μl of 2× CED-3 reaction buffer. - Incubate at 30 °C for 1 h.
- Add 180 μl of distilled water.
- Measure the fl uorescent intensity using a UV spectrophotom-
eter: Excitation 360, Emission 460.
3.1 Purifi cation of
Recombinant CED-3
3.2 Measuring
the Activity of
Recombinant CED-3
Eui Seung Lee and Ding Xuehttp://www.ebook3000.com