Figure 6
Baculovirus expression in insect cells. pFastBac derivatives pCoofy27 (His6), pCoofy28 (His6GST) or pCoofy29 (His6MBP) were transformed into DH10Bac E.coli cells to generate recombinant Baculovirus (Bac-to-Bac®, Life Technologies). HighFive or SF9 cells were either infected with P2 virus stock or Baculo Infected Insect Cells BIIC at dilutions indicated. Cells were harvested after 72h and lyzed in SDS Lämmli buffer. Total lysate was loaded on 4–12% NuPAGE gels (Life Technologies). ø cells infected with non-recombinant Baculovirus. (A) Expression of His6GST-Vasp (lanes 1 to 5) and His6MBP-Vasp (lanes 6 to 10) (UniProt: P50552) in HighFive cells after infection with P2 virus stock; 48 h expression , 2000 fold virus dilution (lanes 1 and 6); 72 h expression at 20.000 (lanes 2 and 7), 2000 (lanes 3 and 8), 200 (lanes 4 and 9) and 40-fold (lanes 5 and 10) virus dilution, respectively; coomassie stained (B) Expression of His6-ODC (NCBI RefSeq NP_002530) in HighFive cells after infection with BIIC; lanes 1–4 BIIC at 1:500, 1:1000, 1:2000, 1:4000 dilution respectively; coomassie stained (C) Expression of Topoisomerase I (UniProt P04786) fused to His6GST-topisomerase I (lanes 1 to 4) and His6MBP (lanes 5 to 7) in High Five cells. Cells were infected with BIIC at 1:500, 1:1000, 1:2000 (lanes 1 to 3 and 5 to 7), and 1:4000 (lane 4) respectively. ø1 cells infected with insertless virus; ø2 uninfected H5 cells, ø3 uninfected SF9 cells, detected by Western Blot analysis using His-Probe™ reagent (ThermoScientific, Braunschweig, Germany).