Efficient production of an antibody Fab fragment using the baculovirus-insect cell system.
The production of an Fab fragment of the catalytic antibody 6D9 in lepidopteran insect cells infected with a recombinant baculovirus that contained both the heavy chain (Hc) and light chain (Lc) genes of the Fab fragment was investigated. Western blotting and enzyme-linked immunosorbent assay (ELISA) of culture supernatant showed that baculovirus-infected Trichoplusia ni BTI-TN-5B1-4 (High Five) cells secreted an Fab fragment that retained antigen-binding activity. Infection of High Five cells with a recombinant baculovirus, in which the Lc and Hc genes were located downstream of the baculovirus p10 and polyhedrin promoters, respectively, produced a higher Fab fragment yield than that obtained with a baculovirus in which the Hc and Lc genes were downstream of the p10 and polyhedrin promoters, respectively. Baculovirus-infected High Five cells secreted more of the Fab fragment than Spodoptera frugiperda Sf9 cells. Moreover, use of the baculovirus gp64 signal sequence upstream of the Lc and Hc genes resulted in greater yield of the secreted Fab fragment than use of the insect-derived BiP and melittin signal sequences. Consequently, the Fab fragment was obtained in a high yield (>600 μg/ml) in a shake-flask culture of High Five cells infected at a multiplicity of infection (MOI) of 10 plaque-forming units (pfu)/cell with the recombinant baculovirus in which the Lc and Hc genes with the gp64 signal sequence were expressed under the control of the p10 and polyhedrin promoters, respectively. These results indicate that the baculovirus-insect cell system may allow efficient production of antibody Fab fragments.
Furuta
Takanori
T
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan.
Ogawa
Takafumi
T
Katsuda
Tomohisa
T
Fujii
Ikuo
I
Yamaji
Hideki
H
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D050438:Genes, Immunoglobulin Heavy Chain; D050439:Genes, Immunoglobulin Light Chain; D005818:Genetic Engineering; D005822:Genetic Vectors; D007140:Immunoglobulin Fab Fragments / Q000096:biosynthesis* / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D018411:Spodoptera / Q000235:genetics* / Q000821:virology*
D007140:Immunoglobulin Fab Fragments; D021382:Protein Sorting Signals; D011994:Recombinant Proteins