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

다국어 초록 (Multilingual Abstract)

The 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.

참고문헌 (Reference)

1 Deparis V, "Use of flow cytometry to monitor infection and recombinant human alpha-1,3/4 fucosyltransferase production in baculovirus infected Sf9cell cultures" 19 : 624-630, 2003

2 Radford KM, "The indirect effects of multiplicity of infection on baculovirus expressed proteins in insect cells:secreted and non-secreted products" 24 : 73-81, 1997

3 Chae‐Yeon Lim, "Purification of anti‐colorectal cancer monoclonal antibody CO17‐1A from insect cell culture using a French press and sonication" 한국곤충학회 45 (45): 102-109, 2015

4 Neutra R, "Optimization of proteinproduction by the baculovirus expression vector system in shake flasks" 37 : 74-78, 1992

5 Park DY, "Optimization of expression conditions for production of anti-colorectal cancer monoclonal antibody CO17-1A in baculovirus-insect cell system" 30 : 419-426, 2011

6 Deuk‐Su Kim, "Optimization of colorectal cancer vaccine candidate protein GA733‐Fc expression in a baculovirus–insect cell system" 한국곤충학회 45 (45): 39-48, 2015

7 Jeong‐Hwan Lee, "Murine response studies of insect cell (Sf9) expressed recombinant colorectal cancer vaccine candidate using surface plasmon resonance studies" 한국곤충학회 46 (46): 5-14, 2016

8 Zaloudik J, "Inhibition of tumor growth by recombinant vaccinia virus expressing GA733/CO17-1A/EpCAM/KSA/KS1-4 antigen in mice" 9 : 382-389, 2002

9 Solá RJ, "Glycosylation of therapeutic proteins:an effective strategy to optimize efficacy" 24 : 9-21, 2010

10 Yo Seph Park, "Functionality of insect‐cell‐derived colorectal cancer vaccine candidate protein EpCAM‐Fc in human dendritic cells" 한국곤충학회 45 (45): 162-166, 2015

11 Se‐Ra Park, "Expression, glycosylation and function of recombinant anti‐colorectal cancer mAb CO17‐1A in SfSWT4 insect cells" 한국곤충학회 44 (44): 39-46, 2014

12 Jeong‐Hwan Lee, "Expression of recombinant anti‐breast cancer immunotherapeutic monoclonal antibody in baculovirus–insect cell system" 한국곤충학회 44 (44): 207-214, 2014

13 Lu Z, "Expression of GA733-Fc fusion protein as a vaccine candidate for colorectal cancer in transgenic plants" 364240 : 1-11, 2012

14 Lim CY, "Effect of the developmental stage and tissue position on the expression and glycosylation of recombinant glycoprotein GA733-FcK in transgenic plants" 5 : 778-, 2015

15 Lu Qiao, "Characterization of the Glycan Structures of Glycoprotein GA733-Fc Expressed in a Baculovirus-Insect Cell System" 대한화학회 36 (36): 139-149, 2015

16 Song MR, "Characterization of Nglycan structures and bio function of anti-colorectal cancer monoclonal antibody CO17-1A produced in baculovirus insect cell expression system" 110 : 135-140, 2010

17 Malde V, "Calculation of baculovirus titer using a microfluidic-based bioanalyzer" 36 : 946-, 2004

18 Strassburg CP, "Baculovirus recombinant expressing a secreted form of a transmembrane carcinoma-associated antigen" 52 : 815-821, 1992

19 Kost TA, "Baculovirus as versatile vectors for protein expression in insect and mammalian cells" 23 : 567-575, 2005

20 Janakiraman V, "A rapid method for estimation of baculovirus titer based on viable cell size" 132 : 48-58, 2006

21 Mulvania T, "A flow cytometric assay for rapid, accurate determination of baculovirus titer" 3 : 47-53, 2004