Potential of real-time measurement of GFP-fusion proteins
Potential of real-time measurement of GFP-fusion proteins
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DOI：
10.1016/j.jbiotec.2003.10.039
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发表时间：
2004-04-08
影响因子：
4.1
通讯作者：
Jenkins, O
中科院分区：
文献类型：
--
作者：
Jones, JJ;Bridges, AM;Jenkins, O
以 Randers-Eichhorn [Biotechnol.生物工程。 55 (1997) 921]，开发了一种用于监测绿色荧光蛋白（GFP）的在线、实时、耐用、可蒸汽灭菌的光学传感器。构建了融合表达蛋白的通用克隆载体，允许 GFP 和目标蛋白作为融合体表达。培养以 201 规模进行，来自传感器的信号直接传送到生物反应器的控制系统。然后在线测量 GFP 的产生，信号直接与其他控制参数连接，从而允许根据重组蛋白表达直接控制微生物过程。获得在线和离线数据之间的正表达相关性。使用 LC-MS 和酶标仪测定对离线测量的蛋白质积累进行定量。这种传感器在工艺开发的许多方面都有巨大的潜力，我们开发了一个工作系统，可以优化生产条件，例如，将 pH 控制直接与融合蛋白联系起来。结果还表明，与天然构建体相比，GFP 不会改变目标蛋白的培养特性。还讨论了表达为融合体的 GFP 是否影响靶蛋白的溶解度。 (C) 2004 Elsevier B.V. 保留所有权利。
Building on the basic design concepts of Randers-Eichhorn [Biotechnol. Bioeng. 55 (1997) 921], an on-line, real-time robust, steam sterilisable optical sensor for monitoring green fluorescent protein (GFP) has been developed. A general cloning vector for fusion expression proteins was constructed, allowing expression of both GFP and the target protein as a fusion. Cultivations were carried out at the 201 scale with the signal from the sensor being relayed directly to the control system of the bioreactors. The production of GFP was then measured on-line, the signal was interfaced directly with other controlling parameters, thereby allowing the microbial process to be controlled directly based on recombinant protein expression. A positive expression correlation between on-line and off-line data was obtained. Protein accretion measured off-line was quantified using both LC-MS and plate reader assays. The potential of such a sensor for many aspects of process development is considerable and we have developed a working system which allows the optimisation of production conditions, for example, linking pH control directly to the fusion protein. Results are also presented that illustrate GFP does not alter the cultivation characteristics of the target protein when compared to the native construct. Whether GFP expressed as a fusion influences the solubility of the target protein is also discussed. (C) 2004 Elsevier B.V. All rights reserved.