Fritsche Claudia, Sitz Mandy, Weiland Norman, Breitling Reinhard, Pohl Hans-Dieter
Department of Medical Engineering and Biotechnology, University of Applied Sciences Jena, Jena, Germany.
J Basic Microbiol. 2007 Oct;47(5):384-93. doi: 10.1002/jobm.200710111.
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.
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成分的营养培养基中培养宿主生物体。因此，针对新的表达系统——大利什曼原虫，开发了各种营养培养基，并根据其在静态悬浮培养和搅拌培养中的培养条件进行了检测。研究结果是基于酵母提取物和缓冲盐开发出了一种不含血清但含有血红素的培养基。在此我们报告，在另一种培养基YE培养基中，可获得0.103 h⁻¹的高且稳定的比生长速率以及1×10⁹个细胞/毫升的最大细胞密度。对于新开发的培养基，可证明增强型绿色荧光蛋白的成功表达以及培养从搅拌培养向生物反应器的适应性。此外，还建立了一种检测必需有机铁源血红素的分析方法。对血红素的消耗进行了监测，因为血红素是生物过程控制中一个潜在的重要过程参数。基于这些结果，一种改进的表达系统可作为生产重组蛋白的常用细胞培养方法的替代方法。
