Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, 1-1, Rokkodai, Nada, Kobe, 657-8501, Japan.
Cytotechnology. 2003 Nov;43(1-3):3-10. doi: 10.1023/B:CYTO.0000039894.27256.0f.
The production of beta-galactosidase by Sf9 cells infected with recombinant Autographa californica nucleopolyhedrovirus (AcNPV) was investigated in shake-flask culture using two serum-free basal media: Grace's medium and TNM-FH (Grace's medium supplemented with lactalbumin hydrolysate and yeast extract). At the time of infection, cells grown in serum-supplemented TNM-FH were transferred into fresh basal media without adaptation. The absence of serum depressed the beta-galactosidase yield considerably in Grace's medium, but to a much lesser extent in TNM-FH, where it reached around 2/3 of the level obtained in TNM-FH supplemented with 10% fetal bovine serum (FBS). While both lactalbumin hydrolysate and yeast extract promoted beta-galactosidase production, their removal by medium replacement on post-infection day 1 gave a beta-galactosidase yield nearly equal to that obtained in their continuous presence. Supplementation of basal media with phosphatidic acid (PA) from egg yolk lecithin, which has been shown to enhance cell growth and recombinant protein production in serum-free culture of Chinese hamster ovary (CHO) cells, was also effective in increasing beta-galactosidase yield. Elevating the multiplicity of infection (MOI) from 2 to 10 plaque-forming units per cell (pfu/cell) also resulted in an increase in product yield. These results provide information important to the development of cost-effective serum-free culture technology for use in large-scale production of recombinant proteins by the baculovirus-insect cell system.
在摇瓶培养中，使用两种无血清基础培养基：Grace 培养基和 TNM-FH（Grace 培养基中添加乳白蛋白水解物和酵母提取物），研究了感染重组美洲棉铃象鼻虫核多角体病毒（AcNPV）的 Sf9 细胞产生β-半乳糖苷酶的情况。在感染时，在含血清的 TNM-FH 中生长的细胞未经适应就转移到新鲜的基础培养基中。在 Grace 培养基中，无血清会大大降低β-半乳糖苷酶的产量，但在 TNM-FH 中，其产量仅为添加 10%胎牛血清（FBS）的 TNM-FH 中的 2/3。虽然乳白蛋白水解物和酵母提取物都能促进β-半乳糖苷酶的产生，但在感染后第 1 天通过培养基替换去除它们会导致β-半乳糖苷酶的产量几乎与它们持续存在时相同。在基础培养基中添加蛋黄卵磷脂中的磷脂酸（PA），这已被证明能增强无血清培养的中国仓鼠卵巢（CHO）细胞的细胞生长和重组蛋白的生产，也能有效提高β-半乳糖苷酶的产量。将感染复数（MOI）从 2 提高到 10 个噬菌斑形成单位/细胞（pfu/cell）也会导致产物产量增加。这些结果为开发具有成本效益的无血清培养技术提供了重要信息，该技术可用于杆状病毒-昆虫细胞系统大规模生产重组蛋白。
