title: Expression and purification of Canis interferon α in Escherichia coli using different tags.
abstract: 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±0.47×10(14) and 2.25±0.87×10(12) 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.
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Fang
authors.initials: F
authors.lastname: Yang
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Yingying
authors.initials: Y
authors.lastname: Pan
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Yazhou
authors.initials: Y
authors.lastname: Chen
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Shiming
authors.initials: S
authors.lastname: Tan
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Mingfei
authors.initials: M
authors.lastname: Jin
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China.
authors.forename: Zirong
authors.initials: Z
authors.lastname: Wu
authors.affiliation: School of Life Science, East China Normal University, Shanghai 200241, PR China. Electronic address: jhuang@bio.ecnu.edu.cn.
authors.forename: Jing
authors.initials: J
authors.lastname: Huang
mesh_terms: D000818:Animals; D004285:Dogs; D004926:Escherichia coli / Q000235:genetics*; D005982:Glutathione Transferase / Q000235:genetics; D002479:Inclusion Bodies; D016898:Interferon-alpha / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013879:Thioredoxins / Q000235:genetics
chemical_list: D016898:Interferon-alpha; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D005982:Glutathione Transferase
keywords: Canis interferon α; Escherichia coli; Fusion protein; Inclusion body; Solubility