The plasmid pRSFDuet2-MBP-BRC4
(KanR), encoding a C-terminal fusion of BRC4 to maltose binding protein
(MBP), bearing an N-terminal His6-tag was generously provided
by Dr. L. Pellegrini and M. Longo, Cambridge University.17 Rosetta2 (DE3) cells (CmR) were transformed
with pRSFDuet2-MBP-BRC4. A 10 mL preculture was prepared and used
to inoculate 500 mL of LB with appropriate antibiotics. After incubation
at 37 °C (200 rpm) until an OD600 of 0.6 was reached,
the cells were induced with 400 μM IPTG. After a further 5 h
at 37 °C (200 rpm) the cells were harvested. The pellet was resuspended
in 15 mL of phosphate buffered saline (PBS, pH 7.5) and stored at
−20 °C.
For protein purification, the cells were
thawed, 0.5 μL benzonase added and lysed by sonication. The
solution was centrifuged for 40 min at 6,500 rpm. The supernatant
was loaded on a Ni-NTA (Qiagen) column previously equilibrated in
PBS. The column was washed with 5 bed volumes of 10 mM imidazole,
PBS pH 7.5, and 5 bed volumes of 20 mM imidazole in PBS pH 7.5 prior
to elution with 2 bed volumes of 250 mM imidazole in PBS pH 7.5. The
protein was concentrated with 30 MWCO spin concentrators (Millipore),
while the buffer was exchanged to 20 mM CHES pH 9.5, 100 mM NaCl,
1 mM EDTA. The purified protein was stored at 4 °C.
(KanR), encoding a C-terminal fusion of BRC4 to maltose binding protein
(MBP), bearing an N-terminal His6-tag was generously provided
by Dr. L. Pellegrini and M. Longo, Cambridge University.17 Rosetta2 (DE3) cells (CmR) were transformed
with pRSFDuet2-MBP-BRC4. A 10 mL preculture was prepared and used
to inoculate 500 mL of LB with appropriate antibiotics. After incubation
at 37 °C (200 rpm) until an OD600 of 0.6 was reached,
the cells were induced with 400 μM IPTG. After a further 5 h
at 37 °C (200 rpm) the cells were harvested. The pellet was resuspended
in 15 mL of phosphate buffered saline (PBS, pH 7.5) and stored at
−20 °C.
For protein purification, the cells were
thawed, 0.5 μL benzonase added and lysed by sonication. The
solution was centrifuged for 40 min at 6,500 rpm. The supernatant
was loaded on a Ni-NTA (Qiagen) column previously equilibrated in
PBS. The column was washed with 5 bed volumes of 10 mM imidazole,
PBS pH 7.5, and 5 bed volumes of 20 mM imidazole in PBS pH 7.5 prior
to elution with 2 bed volumes of 250 mM imidazole in PBS pH 7.5. The
protein was concentrated with 30 MWCO spin concentrators (Millipore),
while the buffer was exchanged to 20 mM CHES pH 9.5, 100 mM NaCl,
1 mM EDTA. The purified protein was stored at 4 °C.