Baculovirus titration method based on MOI values for optimizing recombinant protein expression of the anti‐cancer vaccine candidate GA733‐Fc using Sf9 insect cells

Abstract

AbstractThe baculovirus expression system has been considered as a highly efficient tool for the production of recombinant biopharmaceutical proteins. The recombinant antigenic glycoprotein GA733 is a cell surface protein that is strongly expressed in human colorectal cancer. Efficient virus titration should be established to achieve optimal multiplicity of infection (MOI) conditions, which are in turn essential for strong expression of the recombinant GA733 fused to the human immunoglobulin IgG 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‐Fc expression cassette under the control of the baculovirus polyhedron promoter. MOI values (0.05, 0.1, 0.5, 1, and 3) were calculated based on both microscope observations and results of titration assay and then used to determine the optimum recombinant expression and harvested sample [cell culture media (CM) or cell lysate (CL)]. The pFastBac dual vector carrying the GA733‐Fc gene was constructed to express GA733‐Fc and used to generate recombinant baculoviruses. Western blotting results showed that recombinant protein expression was dependent on the MOI. In addition, CM and CL showed significant differences in protein synthesis and protein secretion capacities. Our findings suggested that our proposed titration method can be used for reliable calculation of MOI values, which significantly influence recombinant GA733‐Fc protein expression in the baculovirus‐insect cell system.