Optimization of growth methods and recombinant protein production in BTI-Tn-5B1-4 insect cells using the baculovirus expression system.
A novel insect cell line from Trichoplusia ni, BTI-Tn 5B1-4 (Tn 5), was compared to Spodoptera frugiperda, Sf 9, cells for production of two recombinant secreted proteins: truncated Epstein-Barr viral attachment protein (EBV gp105) and truncated, soluble tissue factor (sTF). Under optimum conditions for both cell lines, Tn 5 cells produced 28-fold more secreted sTF than Sf 9 cells, respectively, on a per cell basis. The total production of gp105 was similar for the two cell lines. However, Tn5 cells secreted gp105 much more efficiently, resulting in 5-fold higher levels in the extracellular medium. Despite these increases, Tn 5 cells are attachment-dependent, and protein production is sensitive to the cell density (cells/cm2), unlike the Sf9 cell line which can be easily grown and scaled up in cell suspension cultures without significantly affecting its per cell production. Thus, protein production from Tn 5 cells above 0.1 L scales was optimized with respect to cell density using standard techniques for the growth of attachment-dependent cells. Roller bottles precoated with DEAE-based microcarriers and suspension cultures employing collagen-coated microcarriers were found to be effective ways of culturing Tn 5 cells. Predetermined optimal cell densities were used to produce EBV gp105 in microcarrier-coated roller bottles or in suspension cultures using collagen-coated microcarriers at concentrations close to those observed in tissue culture flasks.
Wickham
T J
TJ
Scripps Research Institute, La Jolla, California 92037.
Nemerow
G R
GR
D000956:Antigens, Viral / Q000096:biosynthesis; D016367:Baculoviridae / Q000235:genetics*; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013925:Thromboplastin / Q000096:biosynthesis; D014759:Viral Envelope Proteins / Q000096:biosynthesis; D014763:Viral Matrix Proteins / Q000096:biosynthesis; D001616:beta-Galactosidase / Q000378:metabolism*
D000956:Antigens, Viral; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins; D014763:Viral Matrix Proteins; D013925:Thromboplastin; D001616:beta-Galactosidase