Characterization of the growth behavior of Leishmania tarentolae -: a new expression system for recombinant proteins
Characterization of the growth behavior of Leishmania tarentolae -: a new expression system for recombinant proteins
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DOI：
10.1002/jobm.200710111
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发表时间：
2007-10-01
影响因子：
3.1
通讯作者：
Pohl, Hans-Dieter
中科院分区：
文献类型：
--
作者：
Fritsche, Claudia;Sitz, Mandy;Pohl, Hans-Dieter
用于人类治疗的重组蛋白的生物技术生产需要在不含动物物质的营养培养基中培养宿主生物体。因此，根据其培养条件，如静态悬浮培养和搅拌培养，开发了用于新表达系统的各种营养培养基并进行了检查。调查导致在开发的无血清，但含氯化血红素培养基，酵母提取物和缓冲盐的基础上。本文报道了在另一种培养基（YE-培养基）中获得了0.103 h（-1）的高而稳定的比生长速率和1 × 10（9）个细胞/ml（-1）的最大细胞密度。对于新开发的培养基，可以显示增强型绿色荧光蛋白的成功表达以及培养从搅拌培养到生物反应器的适应性。此外，建立了一种检测必需的有机铁源氯化血红素的分析方法。监测氯化血红素的消耗量，因为氯化血红素是生物工艺控制的潜在重要工艺参数。了解这些结果后，可获得一种改进的表达系统作为生产重组蛋白的常用细胞培养物的替代物。
Biotechnological production of recombinant proteins for human therapy requires a cultivation of the host organism in a nutrient medium free of animal substances. Therefore, various nutrient media for the new expression system Leishmania tarentolae were developed and examined according to their cultivation conditions as static suspension culture and agitated culture. Investigations resulted in the development of a serum-free but hemin containing medium, based on yeast extract and buffer salts. Here we report that a high and stable specific growth rate of 0.103 h(-1) and a maximal cell density of 1 x 10(9) cells ml(-1) is obtained in an alternative medium, the YE-medium. For the newly developed medium, the successful expression of enhanced green fluorescent protein and the adaptation of the cultivation from the agitated culture to the bioreactor could be shown. Furthermore, an analytical method for detection of the essential, organic iron source hemin was established. The consumption of hemin was monitored because hemin is a potentially important process parameter for bioprocess control. With knowledge of these results, an improved expression system is available as an alternative to commonly used cell cultures for the production of recombinant proteins.