Baculovirus titration method based on MOI values foroptimizing recombinant protein expression of the anti-cancervaccine candidate GA733-Fc using Sf9 insect cells
2018
Ghislain MOUSSAVOU, Incheon National University, Incheon, Republic of Korea | Lee, J.H., Chung-Ang University, Seoul, Republic of Korea | Lu QIAO, Seoul National University, Seoul, Republic of Korea | Noh, Y.H., Chung-Ang University, Seoul, Republic of Korea | Shin, Y.K., Chung-Ang University, Seoul, Republic of Korea | Lee, T.J., Chung-Ang University, Seoul, Republic of Korea | Lee, S.H., Incheon National University, Incheon, Republic of Korea | Ko, K., Chung-Ang University, Seoul, Republic of Korea
The baculovirus expression system has been considered as a highly efficient tool forthe production of recombinant biopharmaceutical proteins. The recombinantantigenic glycoprotein GA733 is a cell surface protein that is strongly expressed inhuman colorectal cancer. Efficient virus titration should be established to achieveoptimal multiplicity of infection (MOI) conditions, which are in turn essential forstrong expression of the recombinant GA733 fused to the human immunoglobulinIgG Fc fragment (GA733-Fc) in the baculovirus-insect system. In the present study,the Sf9 cell line was transfected with plasmid DNA containing the GA733-Fcexpression cassette under the control of the baculovirus polyhedron promoter. MOIvalues (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscopeobservations and results of titration assay and then used to determine the optimumrecombinant expression and harvested sample [cell culture media (CM) or cell lysate(CL)]. The pFastBac dual vector carrying the GA733-Fc gene was constructed toexpress GA733-Fc and used to generate recombinant baculoviruses. Western blottingresults showed that recombinant protein expression was dependent on the MOI. Inaddition, CM and CL showed significant differences in protein synthesis and proteinsecretion capacities. Our findings suggested that our proposed titration method can beused for reliable calculation of MOI values, which significantly influencerecombinant GA733-Fc protein expression in the baculovirus-insect cell system.
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