Expression Purification And Anti-viral Activity Determination Of Elastin-like Polypeptide-fused Canine Interferons

Interferons (IFNs) are a class of small proteins with anti-viral, anti-tumor and immune regulatory activities, and canine recombiant IFN-a is commonly used to treat the viral infections with satisfactory therapeutic effects. However, all of the market available products are expressed as His-tagged proteins in E. coli, which require denaturation, renaturation and/or affinity purification. In addition, these products are not only expensive, but have limited in vivo half-lives as well. The present strategies for extension of IFN half-lives include pegylation and fusion with large molecules such as albumin. The former has the difficulty in control of reaction and product quality and the later still requres affinity purification. Elastin-like polypeptides (ELPs) are a class of synthetic pentapolymers with temperature-sensitive reverse transition prpperty and their fusion proteins can be purified by simple methods such as temperature-controlled centrifugation. In addition, ELPs have been used as in vivo drug delievry vectors due to their excellent histocompatibility and slow releasing effect.Canine interleukin-29 (CaIL-29) has the anti-viral activity similar to CaIFN-a, but its genetic product is not available in market. In this study, E. coli codon-adapted coding sequences for the mature peptides of CaIL-19 and CaIFN-al were synthesied and cloned individually into ELP fusion expression vector pET-ELP. The recombinant vector was called pCaIL29-ELP or pELP-CaIFNa. The similarly synthesized coding sequence for CaIFN-y was inserted into pELP-CaIFNa vector and the resultant vector was called pELP-CaIFNay. The three vectors were transformed individually into BL21 (DE3) or BLR (DE3) E. coli and the expression of fusion proteins was induced with IPTG. SDS-PAGE analysis showed that CaIL29-ELP fusion protein was expressed as inclusion bodies, and ELP-CaIFNa and ELP-CaIFNay were expressed as soluble proteins. Then, CaIL29-ELP was expressed as soluble protein at 26℃. The optimized conditions for soluble expression of CaIL29-ELP, ELP-CaIFNa and ELP-CaIFNay were induction for 24 h with 0.2mM IPTG.After expression under optimized conditions, the transition temperatures and NaCl concentrations for purification of CaIL29-ELP, ELP-CalFNa and ELP-CaIFNay by inverse transition cycling (ITC) were optimized to be 26℃,26℃ and 39℃ with final concentrations of NaCl of 3mol/L,3mol/L and 3mol/L, respectively. After dissolving with 6M urea and dialysis against PBS, SDS-PAGE analysis showed that CaIL29-ELP, ELP-CaIFNa and ELP-CaIFNay were purified to 74%,79% and 82% with concentrations of 0.08mg/ml,0.14mg/ml and 0.13mg/ml, respectively. By using MDCK/VSV testing sytem, the anti-viral activities of three fusion CaIFNs were determined to be 2.5 ×104 U/mg,1.4×105 U/mg and 8.4×103 U/mg, respectively. These experimental data suggest that the ELP fusion CalFNs CaIL29-ELP, ELP-CaIFNa and ELP-CaIFNay had higher purities and anti-viral activities with potential extended half-lives and therapeutic effects for treating canine viral infections.