OPTIMIZATION OF GROWTH METHODS AND RECOMBINANT PROTEIN-PRODUCTION IN BTI-TN-5B1-4 INSECT CELLS USING THE BACULOVIRUS EXPRESSION SYSTEM
OPTIMIZATION OF GROWTH METHODS AND RECOMBINANT PROTEIN-PRODUCTION IN BTI-TN-5B1-4 INSECT CELLS USING THE BACULOVIRUS EXPRESSION SYSTEM
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DOI：
10.1021/bp00019a004
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发表时间：
1993-01-01
影响因子：
2.9
通讯作者：
NEMEROW, GR
中科院分区：
文献类型：
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作者：
WICKHAM, TJ;NEMEROW, GR
用一种新的毛癣虫（Trichoplusia ni）昆虫细胞系BTI-Tn 5B1-4 （Tn 5）与frugiperda Spodoptera Sf 9细胞进行比较，发现两种重组分泌蛋白：截断的Epstein-Barr病毒附着蛋白（EBV gp105）和截断的可溶性组织因子（sTF）。在两种细胞系的最佳条件下，以每个细胞为基础，Tn 5细胞分泌的sTF分别是Sf 9细胞的28倍。gp105的总产量在两种细胞系中相似。然而，Tn5细胞更有效地分泌gp105，导致细胞外培养基中的gp105水平高出5倍。尽管有这些增加，但Tn 5细胞是附着依赖性的，蛋白质产量对细胞密度（细胞/平方厘米）很敏感，不像Sf9细胞系可以很容易地在细胞悬浮培养中生长和扩大，而不会显著影响其每个细胞的产量。因此，利用附着依赖性细胞生长的标准技术，对0.1 L以上规模的Tn 5细胞的蛋白质产量进行了优化。采用deae微载体预包被滚瓶和胶原包被微载体悬浮培养是培养Tn - 5细胞的有效方法。预先确定的最佳细胞密度用于在微载体包覆的滚轮瓶中生产EBV gp105，或在接近组织培养瓶中观察到的浓度下使用胶原包覆的微载体进行悬浮培养。
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.