Expression and serological application of recombinant epitope-repeat protein carrying an immunodominant epitope of Newcastle disease virus nucleoprotein

Gaikwad, Satish S.; Lee, Hyun-Jeong; Kim, Ji-Ye; Choi, Kang-Seuk

SF9 suspension cultures (2×10 6 cells/mL) grown in 2 L of Sf900 II medium supplemented with 1% of FBS were infected with recombinant baculovirus at multiplicity of infection of 5 plaque-forming unit/cell. Cells were grown for 72 hours and cell pellets were washed in phosphate buffered saline (PBS it was purified by using Ni-NTA spin columns under denaturant condition with 8 M urea according to Ni-NTA spin handbook (Qiagen, Hilden, Germany). The collected cells were lysed in 10 mL buffer B (10 mM NaH2PO4, 300 mM NaCl, 8 M urea, pH 8.0) and centrifuged the lysate at 10,000×g for 30 minutes and collecting supernatant. Loading up to 600 μL of the cleared lysate supernatant containing the 6× His-tagged protein onto a pre-equilibrated Ni-NTA spin column, centrifuged 2 minutes at 700×g and washing the column with 600 μL buffer C (10 mM NaH2PO4, 300 mM NaCl, 8 M urea, pH 6.3) and centrifuged 2 minutes at 700×g, the recombinant protein was finally eluted with buffer E (10 mM NaH2PO4, 300 mM NaCl, 8 M urea, pH 4.3). The recombinant protein was allowed to refold using refolding buffer (50 mM Tris pH 7.5, 0.5 M NaCl, 0.3% CHAPS, 1 mM DTT, 5% glycerol). rERP was dialyzed against refolding buffer (40 mM CHAPS and 10 mM DTT).