State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), Shanghai, 200237, China.
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai Collaborative Innovation Center for Biomanufacturing (SCICB), Shanghai, 200237, China.
J Biotechnol. 2020 Jan 10;307:131-138. doi: 10.1016/j.jbiotec.2019.11.006. Epub 2019 Nov 6.
The soluble expression of recombinant proteins in Escherichia coli is vital for protein applications in biotechnology and pharmaceuticals. However, the use of E. coli for efficient heterologous protein expression is hampered by several factors, such as poor expression and protein aggregation. Changing the culture or purification conditions may alleviate these issues, but methods based on gene fusion technology offer unique opportunities to improve the production and purification of soluble proteins. Here, we develop a novel fusion tag based on Spy, a newly identified molecular chaperone that functions in the periplasm of E. coli in an ATP-independent manner to prevent protein aggregation and assist in protein folding. We found that the tandem fusion of Spy stands among the well-described best fusion partners, such as MBP and SUMO, in increasing the soluble steady-state levels of six heterologous passenger proteins. Moreover, an easily aggregated passenger protein remained soluble after the removal of the Spy tag, implying that chaperone-dependent folding occurred when the passenger protein was fused to Spy. Our work expands the toolkit of fusion tags and allows them to aid in the production of unstable proteins with industrial or clinical values.
重组蛋白在大肠杆菌中的可溶性表达对于生物技术和制药领域的蛋白质应用至关重要。然而，大肠杆菌在高效异源蛋白表达方面的应用受到多种因素的限制，例如表达水平低和蛋白聚集。改变培养或纯化条件可能会缓解这些问题，但基于基因融合技术的方法提供了独特的机会，可以改善可溶性蛋白的生产和纯化。在这里，我们开发了一种基于 Spy 的新型融合标签，Spy 是一种新发现的分子伴侣，它在大肠杆菌的周质中以非 ATP 依赖的方式发挥作用，以防止蛋白聚集并协助蛋白折叠。我们发现，Spy 的串联融合在增加六种异源载体蛋白的可溶性稳态水平方面，与其他公认的最佳融合伴侣（如 MBP 和 SUMO）相当。此外，在去除 Spy 标签后，一个易于聚集的载体蛋白仍然保持可溶性，这意味着当载体蛋白与 Spy 融合时，发生了伴侣蛋白依赖的折叠。我们的工作扩展了融合标签的工具包，并允许它们帮助生产具有工业或临床价值的不稳定蛋白。
