State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, 155 Yangqiao Road West, Fuzhou, 350002, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Microb Cell Fact. 2022 Apr 2;21(1):51. doi: 10.1186/s12934-022-01776-7.
Producing large amounts of soluble proteins from bacteria remains a challenge, despite the help of current various solubilizing fusion tags. Thus, developing novel tags is necessary. Antifreeze protein (AFP) has excellent solubility and hydrophilicity, but there are no current reports on its use as a solubilizing fusion tag. Additionally, there is no precedent for using retro-proteins (reverse sequence) as solubilizing fusion tags. Therefore, we selected the antifreeze protein AXX and obtained its retro-protein XXA by synthesizing the XXA gene for the development of a new solubilizing fusion tag.
XXA exhibits better stability and ease of expression than AXX; hence, we focused the development of the solubilizing fusion tag on XXA. XXA fused with the tested inclusion bodies, significantly increasing the soluble expression compared with commonly used solubilizing fusion tags such as GST, Trx, Sumo, MBP, and NusA. The tested proteins became soluble after fusion with the XXA tag, and they could be purified. They maintained a soluble form after XXA tag removal. Finally, we used enzymatic digestion reaction and western blot experiments to verify that bdNEDP1 and NbALFA, which were soluble expressed by fusion with XXA, were active.
We developed the novel solubilizing fusion tag XXA, which could more effectively facilitate the soluble expression of inclusion bodies compared with current commonly used tags. XXA could function at both low and high temperatures, and its moderate molecular weight has a limited impact on the output. These properties make XXA an ideal fusion tag for future research and industrial production. Moreover, for the first time, we highlighted the broad potential of antifreeze protein as a solubilizing fusion tag, bringing retro-protein into practical application.
尽管目前有各种可溶性融合标签的帮助，但从细菌中大量生产可溶性蛋白质仍然是一个挑战。因此，有必要开发新的标签。抗冻蛋白（AFP）具有极好的溶解性和亲水性，但目前尚无将其用作可溶性融合标签的报道。此外，没有将反向蛋白（逆序）用作可溶性融合标签的先例。因此，我们选择了抗冻蛋白 AXX，并通过合成 XXA 基因获得了其反向蛋白 XXA，以开发新的可溶性融合标签。
XXA 比 AXX 具有更好的稳定性和表达容易性；因此，我们专注于开发可溶性融合标签 XXA。XXA 与测试包涵体融合，与 GST、Trx、Sumo、MBP 和 NusA 等常用可溶性融合标签相比，可溶性表达显著增加。测试蛋白与 XXA 标签融合后变为可溶性，可进行纯化。在去除 XXA 标签后，它们仍保持可溶性形式。最后，我们使用酶消化反应和 Western blot 实验验证了 bdNEDP1 和 NbALFA，它们通过与 XXA 融合可溶性表达，是有活性的。
我们开发了新型可溶性融合标签 XXA，与目前常用的标签相比，它可以更有效地促进包涵体的可溶性表达。XXA 可以在低温和高温下发挥作用，其适中的分子量对产量的影响有限。这些特性使 XXA 成为未来研究和工业生产的理想融合标签。此外，我们首次强调了抗冻蛋白作为可溶性融合标签的广泛潜力，将反向蛋白引入实际应用。
