Glutathione sepharose

Recombinant glutathione-S-transferase (GST) and GST-fused mutant of Raf kinase inhibitory protein (RKIP-TV-delRBD) were purified from E. coli using glutathione sepharose (Amersham Biosciences). RKIP-TV-delRBD possesses a single mutation of threonine residue 153 to valine (Corbit et al., 2003 (link)) and a deletion mutation of amino acids 77–108, which include a main site for its association with Raf-1 (Yeung et al., 2000 (link)). Purified proteins were dialyzed in HEPES buffered solution (20 mM HEPES, pH 7.2), concentrated by centrifugal filter devices (Millipore), and stored at −80°C.

GST fusion proteins were purified from the Escherichia coli BL-21 strain in lysis buffer using glutathione-Sepharose (Amersham Bioscience). Sepharose beads with GST fusion proteins where incubated with the indicated HEK-293 T-transfected cell lysate in binding buffer O/N at 4 °C. Beads were washed with binding buffer and proteins were eluted with LDS reducing sample buffer (Bio-Rad) by heating at 70 °C.
Lysis buffer: 20 mM Tris-HCl, pH 7.4, 1 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol, 1 mg ml⁻¹ lysozyme, 1 mM PMSF and a protease inhibitor tablet (Roche).
Binding buffer: 25 mM Tris-HCl, pH 7.5, 200 mM NaCl, 0.2% Nonidet P-40, 1 mM DTT, 1 mM EDTA, 10% Glycerol and a proteases inhibitor tablet (Roche).

Recombinant proteins were produced in BL21-DE3 E. coli strains. Protein expression was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside for 16 hr at 18°C. Bacterial pellets were lysed in either His buffer (100 mM HEPES [pH 7.5], 300 mM NaCl) or GST buffer (TBS; 50 mM Tris [pH 7.5], 150 mM NaCl, 2 mM dithiothreitol) supplemented with protease inhibitors. Proteins were purified with nickel agarose (QIAGEN) or glutathione Sepharose (Amersham Biosciences). For microinjection, protein stocks were diluted with TBS to the indicated concentrations.

Guinea pig anti-NS1 [15 (link)] and rabbit anti-NP antibodies [46 (link)] have been previously described. Rhodamine Red X-labeled anti-guinea pig immunoglobulins and FITC-labeled anti-rabbit immunoglobulins were used as secondary antibodies in immunofluorescence analysis (1:100, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). In Western blotting guinea pig anti-NS1 [15 (link)], rabbit anti-Actin (sc-10731; 1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-MxA [47 (link)], rabbit anti-p-IRF3 (#4947; 1:1000 dilution: Cell Signaling Technology, Inc., Beverly, MA, USA) and rabbit anti-p-Akt (#9271 s; 1:1000 dilution: Cell Signaling Technology,) immunoglobulins were used. For anti-M1 and anti-NEP antibodies, Glutathione S-transferase (GST) –tagged A/Udorn/72 GST-M1 and polyhistidine-tagged A/Udorn/72 His-NEP (aa 11–121) were expressed in Escherichia coli, purified with Glutathione-Sepharose (Amersham Biosciences, Buckinghamshire, United Kingdom) or preparative SDS-PAGE, respectively, and used to immunize rabbits four times at 3-week intervals (100 μg or 60 μg of antigen/immunization, respectively). As secondary antibodies in Western blot HRP-conjugated rabbit anti-guinea pig (1:1000 dilution; Dako, Glostrup, Denmark) and goat anti-rabbit (1:2000 dilution; Dako) immunoglobulins were used.

Both catalytic subunits were overexpressed and purified as described previously [44 (link)]. Briefly, E. coli BL21(DE3)trxB cells (Novagen Merck KGaA, Darmstadt, Germany) harboring the plasmid pGEX-3X::hsCK2α or pGEX-3X::hsCK2α’ were grown until OD₆₀₀ = 0.6 at 37 °C. Next, IPTG was added to the final concentration of 0.2 mM and the cultures were continued at room temperature for 4 h and then centrifuged at 5000× g for 10 min. Bacterial cells were disrupted by sonication and the supernatant was purified using glutathione-sepharose (Amersham Pharmacia Biotech UK Ltd., Buckinghamshire, UK). Fractions containing the CK2 subunit were pooled and dialyzed against 50 mM Tris/HCl buffer, pH 7.5, supplemented with 6 mM β-mercaptoethanol and 30% glycerol. The obtained protein preparations were used in enzymatic assays.