BioSilta Oy, c/o Department of Biochemistry, University of Oulu, Finland.
Microb Cell Fact. 2010 Feb 19;9:11. doi: 10.1186/1475-2859-9-11.
Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures.
The enzymatic glucose release system together with a well-balanced combination of mineral salts and complex medium additives provided high cell densities, high protein yields and a considerably improved proportion of soluble proteins in harvested cells. The cultivation method consists of three steps: 1) controlled growth by glucose-limited fed-batch to OD600 approximately 10, 2) addition of growth boosters together with an inducer providing efficient protein synthesis within a 3 to 6 hours period, and 3) a slow growth period (16 to 21 hours) during which the recombinant protein is slowly synthesized and folded. Cell densities corresponding to 10 to 15 g l(-1) cell dry weight could be achieved with the developed technique. In comparison to standard cultures in LB, Terrific Broth and mineral salt medium, we typically achieved over 10-fold higher volumetric yields of soluble recombinant proteins.
We have demonstrated that by applying the novel EnBase Flo cultivation system in shaken cultures high cell densities can be obtained without impairing the productivity per cell. Especially the yield of soluble (correctly folded) proteins was significantly improved in comparison to commonly used LB, Terrific Broth or mineral salt media. This improvement is thought to result from a well controlled physiological state during the whole process. The higher volumetric yields enable the use of lower culture volumes and can thus significantly reduce the amount of time and effort needed for downstream processing or process optimization. We claim that the new cultivation system is widely applicable and, as it is very simple to apply, could widely replace standard shake flask approaches.
摇瓶中的重组蛋白生产需要提供高细胞密度、每细胞高蛋白生产力和良好的蛋白质量。由于氧气耗尽、缺乏 pH 控制以及需要使用低诱导细胞密度，实验室手册中描述的方法往往无法达到这些目标。在本文中，我们描述了一种新型酶控补料分批培养技术对简单摇瓶培养大肠杆菌中重组蛋白生产的影响。
酶控葡萄糖释放系统与矿物质盐和复杂培养基添加剂的平衡组合提供了高细胞密度、高蛋白产量和收获细胞中可溶性蛋白的比例显著提高。该培养方法包括三个步骤：1）通过葡萄糖限制补料分批培养至 OD600 约 10，2）添加生长促进剂和诱导剂，在 3 至 6 小时内实现有效的蛋白合成，3）在缓慢生长阶段（16 至 21 小时）中，重组蛋白缓慢合成和折叠。用开发的技术可以达到 10 至 15 g l(-1) 细胞干重的细胞密度。与 LB、Terrific Broth 和矿物质盐培养基中的标准培养相比，我们通常可以实现超过 10 倍的可溶性重组蛋白的比体积产率。
我们已经证明，通过在摇瓶培养中应用新型 EnBase Flo 培养系统，可以在不损害每细胞生产力的情况下获得高细胞密度。与常用的 LB、Terrific Broth 或矿物质盐培养基相比，可溶性（正确折叠）蛋白的产量显著提高。这种改进被认为是由于整个过程中生理状态得到了很好的控制。更高的比体积产率可以使用更小的培养体积，从而显著减少下游处理或工艺优化所需的时间和精力。我们声称新的培养系统具有广泛的适用性，并且由于它非常易于应用，因此可以广泛替代标准摇瓶方法。
