Purification and Immunization of Recombinant Proteins

To obtain purified recombinant proteins, BL21 (DE3) E. coli transformed with pGEX-UBC9, pGEX-VP55, or pGEX-VP55M were cultivated in Luria-Bertani medium containing 100 μg ampicillin/mL at 37°C until the optical density reached 0.4-0.6 at 600 nm. Expression of the GST fusion proteins was induced using 0.1-0.5 mM IPTG (isopropyl-β-D-thiogalactopyranoside) for 4-6 h. The pellet was collected by centrifugation at 5000× g for 5 min and resuspended in PBS (140 mM NaCl, 2.5 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄). The expressed protein was purified as previously described [42 (link)] or using the Glutathione Sepharose™ 4B kit instructions (GE Healthcare). Purified GST-VP55 was used to prepare polyclonal antiserum. Three New Zealand rabbits were intracutaneously immunized with 250 μg of GST-VP55 in Freud's adjuvant (Sigma, USA) at 2 week intervals for three times. Immunoglobulin (IgG) specific for VP55 was purified using Protein A Agarose (Santa Cruz).