Zulkifly Nurul Asma Hasliza, Selas Castiñeiras Tania, Overton Tim W
School of Chemical Engineering and Institute of Microbiology and Infection, The University of Birmingham, Birmingham, United Kingdom.
Cobra Biologics, Keele, United Kingdom.
Front Bioeng Biotechnol. 2023 Aug 2;11:1171823. doi: 10.3389/fbioe.2023.1171823. eCollection 2023.
is commonly used industrially to manufacture recombinant proteins for biopharmaceutical applications, as well as in academic and industrial settings for R&D purposes. Optimisation of recombinant protein production remains problematic as many proteins are difficult to make, and process conditions must be optimised for each individual protein. An approach to accelerate process development is the use of a green fluorescent protein (GFP) fusions, which can be used to rapidly and simply measure the quantity and folding state of the protein of interest. In this study, we used GFP fusions to optimise production of recombinant human protein tumour necrosis factor (rhTNFα) using a T7 expression system. Flow cytometry was used to measure fluorescence and cell viability on a single cell level to determine culture heterogeneity. Fluorescence measurements were found to be comparable to data generated by subcellular fractionation and SDS-PAGE, a far more time-intensive technique. We compared production of rhTNFα-GFP with that of GFP alone to determine the impact of rhTNFα on expression levels. Optimised shakeflask conditions were then transferred to fed-batch high cell density bioreactor cultures. Finally, the expression of GFP from a p expression vector was compared to the T7 system. We highlight the utility of GFP fusions and flow cytometry for rapid process development.
在工业上通常用于生产生物制药应用的重组蛋白，以及在学术和工业环境中用于研发目的。重组蛋白生产的优化仍然存在问题，因为许多蛋白质难以制备，并且必须针对每种蛋白质优化工艺条件。加速工艺开发的一种方法是使用绿色荧光蛋白（GFP）融合蛋白，它可用于快速简单地测量目标蛋白的数量和折叠状态。在本研究中，我们使用GFP融合蛋白通过T7表达系统优化重组人蛋白肿瘤坏死因子（rhTNFα）的生产。流式细胞术用于在单细胞水平上测量荧光和细胞活力，以确定培养物的异质性。发现荧光测量结果与亚细胞分级分离和SDS-PAGE产生的数据相当，而后者是一种耗时得多的技术。我们比较了rhTNFα-GFP与单独GFP的生产情况，以确定rhTNFα对表达水平的影响。然后将优化的摇瓶条件转移到补料分批高细胞密度生物反应器培养中。最后，将p表达载体中GFP的表达与T7系统进行了比较。我们强调了GFP融合蛋白和流式细胞术在快速工艺开发中的实用性。
