Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system

Abstract

The baculovirus-insect cell expression system has been used to produce functional recombinant proteins. The antigen GA733 is a cell-surface glycoprotein highly expressed on most human colorectal carcinoma cells. Conditions for the expression of GA733 fused to the human immunoglobulin IgG Fc fragment (GA733-Fc) were optimized in the baculovirus expression system. Several variable factors were adjusted to optimize expression, including the cell line (Sf9 and High Five), multiplicity of infection (MOI) value (0.05, 0.1, 0.5, 1 and 3), post-infection time (48, 72 and 96 h) and harvested sample (cell culture media (CM) or cell lysate (CL)). In addition, two pFastBac Dual vectors carrying the GA733-Fc gene were constructed to express GA733-Fc with or without an endoplasmic reticulum (ER) retention sequence KDEL and used to generate recombinant baculoviruses. Western blot showed that expression depended on the conditions used to express the recombinant proteins. The protein production level and secretion capability differed in each cell line. In Sf9 cells, the highest expression in the CM and CL was obtained with GA733-Fc at 96 h post-infection at 0.1 MOI and with GA733-FcK at 96 h post-infection at 3 MOI, respectively. In High Five cells, the highest expression in the CM and CL was obtained with GA733-Fc at 48 h post-infection at 1 MOI and with GA733-FcK at 48 h post-infection at 3 MOI, respectively. These results suggest that the MOI value, post-infection time and subcellular localization affect expression, and that these conditions can be modified to optimize protein expression in the baculovirus-insect cell system.

Purification of anti‐colorectal cancer monoclonal antibody CO17‐1A from insect cell culture using a French press and sonication

“…In previous studies, fusing KDEL at the C terminus of a protein resulted in proteins accumulating in the ER, which in turn, enhanced protein expression levels (Park et al 2011;Lu et al 2012;Kim et al 2015;Qiao et al 2015). In our current study, expression of the mAb CO17-1A fused to KDEL (mAb CO17-1AK) was higher than the mAb Figure 5 Analysis of amino acid sequences on HC and LC of mAb CO17-1A.…”

Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies

“…These results suggest that the ER retention signal KDEL retains GA733-Fc in the ER without secretion to CM (in the absence of the signal peptide, the default secretion pathway was not followed). Attachment of KDEL has been used to enhance production of recombinant proteins with oligomannose glycan structure in animal, plant and insect cells (Henderson et al 1996;Schouten et al 1996;Lee et al 2013;So et al 2013;Kim et al 2015;Lim et al 2015). Oligomannose can enhance an immune response because of the interaction between oligomannose of glycoproteins and the mannose receptor on macrophages and dendritic cells.…”

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

“…The baculovirus expression vector system (BEVS) has been proven useful for the production of recombinant proteins in eukaryotic insect cells (Deparis et al 2003;Song et al 2010;Park et al 2011Park et al , 2014Park et al , 2015Lee & Ko 2014;Kim et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005).…”

“…BEVS has many advantages, including scalability using high-density suspension cultures, capacity for expressing multiple genes at high expression levels, and the possibility of post-translational modifications (Deparis et al 2003;Malde & Hunt 2004;Kost et al, 2005). In the present study, we used the BEVS technology to express diverse recombinant proteins, including anti-colorectal cancer monoclonal antibody CO17-1A (Song et al 2010;Park et al 2011Park et al , 2014Lee & Ko 2014 and anti-cancer vaccine candidate GA733-Fc (Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016).…”

“…Optimization of infection conditions influences the expression of recombinant proteins, such as the monoclonal antibody CO17-1A (Park et al 2011), which recognizes the colorectal antigen GA733-2 (gastrointestinal tumorassociated antigen 2) (Strassburg et al 1992;Zaloudik et al 2002;Lu et al 2012;Kim et al 2015;Park et al 2015;Qiao et al 2015;Lim et al 2015aLim et al , 2015bLee et al 2016). Accurate viral stock titration is crucial for determining the multiplicity of infection (MOI) value, which represents the total number of virions added per cell during infection (Neutra et al 1992;Mulvania et al 2004;Janakiraman et al 2006) and ultimately determines recombinant protein expression (Radford et al, 1997).…”