Retro-protein XXA is a remarkable solubilizing fusion tag for inclusion bodies

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.