Expression and purification of recombinant proteins
His-tagged proteins were expressed in E. coli strain
Rosetta2(DE3). Cells were cultivated in LB medium at 37oC to an optical density between 0.6 and 0.8. After
induction with 1 mM of isopropyl-β-D-thiogalactopyranosid (IPTG) the
cells were grown for additional 3 hours at 30 oC.
Collected cells were resuspended in washing buffer (20 mM HEPES, 300 mM
NaCl, 40 mM imidazole, 2 mM DTT, pH 8.0). Cells were disrupted by
sonification and after centrifugation, the supernatant was either loaded
onto a nickel-nitrilotriacetic acid column (GE Healthcare) or incubated
with nickel-nitrilotriacetic acid resin (Qiagen). After washing with
washing buffer the fusion constructs were eluted with elution buffer (20
mM HEPES, 300 mM NaCl, 250 mM imidazole, 2 mM DTT, pH 8.0).
Subsequently, the imidazole was removed from the buffer using PD-10
columns (GE Healthcare). The protein was kept on ice and used directly
or was stored at -20°C until usage.
GST-Get3b was expressed as described above with the following
modifications. Collected bacterial cells were resuspended in 1x PBS
buffer (140 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.8 mM
KH2PO4, pH 7.3) before sonication.
Proteins were purified using glutathione-sepharose (GE Healthcare) and
eluted with 10 mM reduced glutathione in 1xPBS buffer. The buffer was
exchanged to 1xPBS using PD-10 columns (GE Healthcare).