Expression and purification of Canis interferon α in Escherichia coli using different tags

The potent and broad activity of Canis interferon α (CaIFNα) makes it an attractive candidate for the treatment of many viral diseases of dogs. Here, we fused CaIFNα to three different protein tags: thioredoxin (Trx), glutathione S-transferase (GST), and NusA (Nus), to facilitate its expression and purification in Escherichia coli. The Trx-CaIFNα and GST-CaIFNα fusion proteins formed inclusion bodies, while the Nus-CaIFNα protein was soluble when expressed at low temperatures. Trx-CaIFNα was purified from inclusion bodies and refolded, while Nus-CaIFNα was purified under native conditions. The purity of Trx-CaIFNα and Nus-CaIFNα was greater than 90%, and their yields were 74.8% and 6.5%, respectively. Both Trx-CaIFNα and Nus-CaIFNα had antiviral activity in vitro. Their anti-viral activity was 1.09 ± 0.47 × 10¹⁴ and 2.25 ± 0.87 × 10¹² U/mol, respectively, on Madin-Darby canine kidney cells. Both purification methods had advantages and disadvantages. A greater amount of Trx-CaIFNα was obtained, but refolding was required to obtain active protein. In contrast, soluble Nus-CaIFNα did not require refolding, which saved time and materials. However, Nus-CaIFNα, which contained a larger tag, had lower activity than Trx-CaIFNα. In general, we provided two protocols to obtain large amounts of CaIFNα with high antiviral activity. These protocols may promote the clinical development of CaIFNα in treating viral diseases in dog.