Expression, Purification, Anti-viral Activities And In Vitro Half-lives Study Of Canine Interferon-β Using ELP Tag

The viral diseases such as canine parvovirus infection, distemper and viral hepatitis are still prevalent despite the vaccination is commonly applied. Type Ⅰ interferons (IFNs) are a class of cytokines with anti-viral, anti-tumor and immunoregulatory effects and the recombinant IFN-α is commonly used to treat canine viral diseases with good therapeutic effects. The present CaIFN-α products are expressed mostly as His-tagged proteins and inclusion bodies, which require denaturation, renaturation and affinity purification. These products are not only expensive to use, but have limited half-lives in vivo as well. The present strategies for extension of IFN half-life include pegylation and fusion expression with albumin. The former method has the difficulty in control of reaction conditions and product qualities. The later method still requires affinity purification. Elastin-like polypeptides (ELPs) are a class of polypeptides synthesized according to the repeat sequences of elastin. The polypeptides and their fusion proteins can undergo temperature-sensitive reverse transition and can be purified by simple methods such as centrifugation. In addition, ELPs have been used as drug delivery vectors to prolong the half-lives in vivo.Canine interferon-P (CaIFN-β) has the similar anti-viral activity as CaIFN-α, but the genetic product is not available in market. In this study, the coding sequence for the mature peptide of CaIFN-β was amplified by PCR and inserted into prokaryotic expression vector pET-30a or ELP fusion expression vector pET-ELP. The recombinant vector pET-CaIFNβ or pCaIFNβ-ELP was transformed into BL21 (DE3) E. coli and the expression of fusion protein was induced with IPTG. SDS-PAGE analysis showed that both His-CaIFNβ and CaIFNβ-ELP fusion proteins were expressed as inclusion bodies at 37℃. The induction temperatures were optimized for soluble expression of two proteins. However, the His-CaIFNβ fusion protein was still expressed as inclusion body even at 20℃. The CaIFNβ-ELP fusion protein was expressed mainly as soluble or insoluble protein below or above 30℃. This indicates that ELP had soluble effect on CaIFN-β. The expression conditions including IPTG concentration and induction time were further optimized for soluble expression of CaIFNβ-ELP fusion protein.These data provided a good foundation for batch purification and anti-viral activity study of the two fusion proteins.The expression of His-CaIFNβ and CaIFNβ-ELP fusion proteins were induced at the optimized conditions. After denaturation and renaturation, the His-CaIFNβ protein was purified by affinity column with a purity of 85% and yield of 50 mg/L. The CaIFNβ-ELP fusion protein was purified by inverse transition cycling (ITC) under the optimized temperature (28℃), NaCl concentration (2.0 M) and centrifugation force (12,000 g). The purified protein was dissolved in 4 M urea and dialyzed against PBS with a purity of 96% and yield of 220 mg/L. By using MDCK/VSV system, the anti-viral assay showed that the ITC-purified CaIFNβ-ELP fusion protein had the anti-viral activity of 105U/mg, which was significantly higher than that (104 U/mg) of affinity-purified His-CaIFNβ fusion protein. The stability assay in mouse plasma showed that CaIFNβ-ELP fusion protein had an in vitro half-life of>24 h, which was significantly longer than that (12 h) of His-CaIFNβ fusion protein. These data suggest that ELP could be used as an efficient tag for expression and purification recombinant CaIFN-β. The purified CaIFNβ-ELP fusion protein had a higher anti-viral activity and extended half-life, and could be used to treat canine virus infections.