Large-scale expression of recombinant human HDACs was carried out
as described previously.32 (link),37 (link) Briefly, HEK293/T17
cells were transiently transfected with expression pMM222 plasmids
comprising N-terminally tagged (TwinStrep-FLAG-HALO tag;Figure S1 ) HDAC genes using linear poly(ethyleneimine)
(PEI, Polysciences Inc.). Cells were harvested three days post-transfection,
and cell pellets were resuspended in the lysis buffer (100 mM Tris-HCl,
10 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 10% glycerol at pH
8.0). Cells were disrupted by sonication, cell lysates were cleared
by centrifugation, and soluble fusion proteins were first purified
by Streptactin affinity chromatography (IBA). For all HDACs but HDACs
1, 10, and 11, the N-terminal tag was cleaved off overnight by the
addition of TEV protease (10:1 HDAC/TEV ratio). Size-exclusion chromatography
on a Superose 6 column (GE Healthcare Bio-Sciences; running buffer
30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP)
was used as the final purification step for all HDAC constructs.
as described previously.32 (link),37 (link) Briefly, HEK293/T17
cells were transiently transfected with expression pMM222 plasmids
comprising N-terminally tagged (TwinStrep-FLAG-HALO tag;
(PEI, Polysciences Inc.). Cells were harvested three days post-transfection,
and cell pellets were resuspended in the lysis buffer (100 mM Tris-HCl,
10 mM NaCl, 5 mM KCl, 2 mM MgCl2, and 10% glycerol at pH
8.0). Cells were disrupted by sonication, cell lysates were cleared
by centrifugation, and soluble fusion proteins were first purified
by Streptactin affinity chromatography (IBA). For all HDACs but HDACs
1, 10, and 11, the N-terminal tag was cleaved off overnight by the
addition of TEV protease (10:1 HDAC/TEV ratio). Size-exclusion chromatography
on a Superose 6 column (GE Healthcare Bio-Sciences; running buffer
30 mM HEPES, 140 mM NaCl, 10 mM KCl, 3% glycerol, and 0.25 mM TCEP)
was used as the final purification step for all HDAC constructs.
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