De Coninck Tibo, Vanhaeren Hannes, Van Damme Els J M
Laboratory for Biochemistry and Glycobiology, Department of Biotechnology, Ghent University, Proeftuinstraat 86, 9000 Ghent, Belgium.
Int J Mol Sci. 2025 Sep 15;26(18):8974. doi: 10.3390/ijms26188974.
Biochemical characterization of proteins is fundamental to understanding their function. Typically, research in protein structure/function requires reasonable quantities of the protein of interest. Because of the low abundances in their natural environment, the heterogenous state of post-translational modifications, and the difficulty of obtaining the tissue containing the protein of interest, recombinant protein production is usually employed. One of the major difficulties impeding advances in biotechnological research is protein insolubility, undermining further downstream research and applications. strains are popular hosts for protein production but are often unfit for the expression of eukaryotic sequences due to the absence of proper post-translational modifications, some of which are crucial for protein folding and activity. Here, we showcase the challenges researchers may be confronted with when trying to produce proteins recombinantly, by using OsAPSE, an enzyme from rice, as an example of a difficult-to-produce protein. Several production hosts were explored, and best results were obtained when OsAPSE was produced in combined with a solubility tag or when a higher eukaryotic system was used. This study highlights common pitfalls in protein research and provides strategies to overcome them, making it a case study for researchers facing similar challenges.
蛋白质的生化特性分析是理解其功能的基础。通常，蛋白质结构/功能的研究需要相当数量的目标蛋白质。由于其在天然环境中的丰度较低、翻译后修饰的异质性状态以及获取含有目标蛋白质的组织的困难，通常采用重组蛋白生产。阻碍生物技术研究进展的主要困难之一是蛋白质不溶性，这会破坏进一步的下游研究和应用。大肠杆菌是常用的蛋白质生产宿主，但由于缺乏适当的翻译后修饰，它们通常不适合表达真核序列，其中一些修饰对蛋白质折叠和活性至关重要。在这里，我们以水稻中的一种酶OsAPSE为例，展示了研究人员在尝试重组生产蛋白质时可能面临的挑战，OsAPSE是一种难以生产的蛋白质。我们探索了几种生产宿主，当OsAPSE与溶解度标签一起生产或使用更高等的真核系统时，获得了最佳结果。这项研究突出了蛋白质研究中常见的陷阱，并提供了克服这些陷阱的策略，使其成为面临类似挑战的研究人员的一个案例研究。
