210
- Incubate the starter cultures for at least 8 h at 37 °C with
constant shaking at ~225 rpm (the cultures can be left to grow
overnight if required). - Add 5 mL of a starter culture to each 1 L culture fl ask and
incubate at 37 °C for 16 h with shaking at ~225 rpm. - Harvest the cells by centrifugation at 5,000 × g for 20 min at
4 °C; discard the supernatant and store each 250 mL cell cul-
ture separately at −20 °C, until required.
A single-step IMAC purifi cation often suffi ces for metacaspase
assays, but the protein must be exchanged into a suitable assay buf-
fer prior to the experiment. For crystallization experiments the
protein should be further purifi ed by size-exclusion
chromatography.- Thaw a 250 mL cell culture ( see Note 13 ) at room tempera-
ture and resuspend in Lysis Buffer using around 5 mL of buf-
fer per 1 g of cultured cells. - Lyse the cells by three passages through a French pressure cell
at 14,000 psi (internal pressure) or equivalent. - Remove cell debris by centrifugation at 48,000 × g for 20 min
at 4 °C. - Collect the clarifi ed supernatant and fi lter-sterilize (0.22 μm).
- Apply the supernatant to a 5 mL HisTrap (IMAC) column
equilibrated in Buffer A with 25 mM imidazole (5 % Buffer B)
and wash for 10 column volumes (CV) in the same buffer. - Wash the column with Buffer A containing 50 mM imidazole
(5 CV, 10 % Buffer B) and elute the protein with 250 mM
imidazole (5 CV, 50 % Buffer B) ( see Note 14 ), collecting
1 mL fractions. - Detect the fractions containing protein using the A 280 trace,
from the protein purifi cation system, and identify which frac-
tions contain the target metacaspase using SDS-PAGE analysis
with a 4–12 % gel and MES Buffer. - Pool the fractions containing the purifi ed protein. NB: If the
protein is to be used solely in activity assays move on to
Subheading 3.5. - Concentrate the protein from the IMAC column to around
3 mL using a centrifugal concentrator with a 30 kDa molecu-
lar weight cutoff. - Sterile fi lter the sample before loading onto an S200 column
equilibrated with 2 CV of GF buffer and run for 1.5 CV in the
same buffer. - Pool all fractions containing the protein.
3.2 Metacaspase
Purifi cation
Karen McLuskey et al.http://www.ebook3000.com