Baculovirus expression system

The human wild-type and mutant versions of BCL6 (Uniprot entry: A5PL18, residue 5–129 or 5–360) and SIAH1 variants (Uniprot Entry: Q8IUQ4, residue 90–282 (substrate binding domain, SBD) or full length), were cloned in pAC-derived vectors³² . Baculovirus for protein expression (Invitrogen) was generated by transfection into Spodoptera frugiperda (Sf9) cells at a density of 0.9 × 10⁶ cells/mL grown in ESF 921 media (Expression Systems), followed by three rounds of infection in Sf9 cells to increase viral titer. Recombinant proteins were expressed and purified as N-terminal His₆ C-terminal Spy (wild-type and mutant versions of BCL6⁵⁻³⁶⁰), N-terminal Strep II-Avi (BCL6⁵⁻¹²⁹, BCL6⁵⁻³⁶⁰, and SIAH1^SBD), and N-terminal Flag (SIAH1^FL) fusions in Trichoplusiani High Five insect cells using the baculovirus expression system (Invitrogen) as described previously³³ .

Protein kinase activity assays and inhibitor screening were performed as previously described (19 (link), 20 (link), 42 , 43 (link)). Briefly, the kinase substrate GST-FLT3S was purified from E. coli cells by using a glutathione-Sepharose column. Recombinant proteins that contained the catalytic domains of wild type and D835H/D835Y mutant forms of human FLT3 (aa573-993), human JAK3 (aa806-1124), and human cKit (aa558-976) were expressed by using the baculovirus expression system (Invitrogen) and isolated from recombinant baculovirus-infected Sf9 insect cells by using the NTA-Ni resin. Phosphorylation of GST-FLT3S by isolated recombinant protein kinases was carried out in a reaction buffer that contained 25 mM Tris–HCl (pH 7.5), 10 mM MgCl₂, 0.2 mM ATP, and 2 mM dithiothreitol in the presence of various concentrations of protein kinase inhibitors. The level of GST-FLT3S tyrosine phosphorylation was determined by immunoblotting with antiphosphotyrosine antibody PY20 followed by horseradish peroxidase-conjugated secondary antibody. Detection and quantification of enhanced chemiluminescence signals were done by using FluorChem SP imaging system from Alpha Innotech (CA, USA).

Human DDB1ΔB and human CRBN were cloned in pAC-derived vectors^{30 (link)} and recombinant proteins were expressed as N-terminal His₆ (DDB1ΔB) or StrepII-Avi (CRBN) fusions in Trichoplusia ni High-Five insect cells using the baculovirus expression system (Invitrogen). For purification, cells were resuspended in buffer containing 50 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) pH 8.0, 200 mM NaCl, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 1 mM phenylmethylsulfonyl fluoride (PMSF), 1x protease inhibitor cocktail (2 μg/ml Aprotinin, 10μM Bestatin, 2μM E-64, 10μM Leupeptin, 1μM Pepstatin A and 10μM 1,10 - Phenanthrolin and 1μM Phosphoramidon) and lysed by sonication. Following ultracentrifugation, the soluble fraction was passed over Strep-Tactin XT (IBA, 2-4030-025) resin and eluted with wash buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM TCEP) supplemented with 50 mM biotin (MedChemExpress, HY-B0511). The affinity-purified protein was subjected to ion exchange chromatography (Poros 50HQ) followed by size exclusion chromatography (16/60 S200, GE) in 50 mM HEPES pH 7.4, 200 mM NaCl and 1 mM TCEP. Biotinylation of was performed as previously described.^{31 (link)} The protein-containing fractions were concentrated using ultrafiltration (Millipore) and flash frozen in liquid nitrogen at 40-120 μM concentration and stored at −80°C.