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Highlights • We expressed three ELP-tagged and two His-tagged PoIFNs in E. col. • We optimized conditions for soluble expression and purification of ELP-tagged PoIFNs. • We compared two ELP fusion orders on PoIFN expression and bioactivity. • We demonstrated the solubility of ELP-fused of PoIFNs under the physical conditions. • We demonstrated that ELP-fusion is a feasible strategy for easy purification and half-life extension of rPoIFNs. Abstract The clinical use of recombinant interferons (rIFNs) is limited by higher purification cost and quick clearance from circulation. Elastin-like polypeptides (ELPs) are a novel tag for recombinant protein purification and half-life extension. In this study, we evaluated the feasibility of ELP fusion for simple purification and half-life extension of recombinant porcine IFNs (rPoIFNs). After construction of five different fusion expression vectors, we optimized the conditions for soluble protein expression and purification. SDS-PAGE analysis showed that, unlike PoIFNα-His and PoIFNγ-His, PoIFNα-ELP, ELP-PoIFNα and PoIFNαγ-ELP were expressed mainly as soluble proteins at 20 ℃. The optimal conditions for the inverse transition cycling (ITC) of three ELP fusion proteins were 2 M NaCl at 28 ℃. After two rounds of ITC, the three ELP fusion proteins were purified to more than 90% purities, which were comparable to that of affinity-purified PoIFNα-His and PoIFNγ-His. Cytopathic effect inhibition assay showed that the five rPoIFNs had potent but different antiviral activities against two different viruses on two different cell types. The plasma solubility assay showed that the three ELP-fused rPoIFNs remained as soluble proteins under the physical conditions. The plasma stability of three ELP-fused rPoIFNs was significantly improved in comparison with that of PoIFN-α. These data suggest that ELP fusion is a feasible strategy to enhance purification and plasma stability of rPoIFNs. [ABSTRACT FROM AUTHOR]

Chicken egg yolk immunoglobulin (IgY) is a superior alternative to mammalian immunoglobulin, but its practical application is limited due to the complex purification procedure. In this study, the C2 domain of streptococcal protein G (SPG) with the binding affinity for chicken IgY was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein, and purified to a high purity by inverse transition cycling (ITC). Binding experiments showed that chicken IgY could bind to and eluted off the ELP-C2 fusion protein in pH-, temperature- and/or time-dependent manners. By using the ELP-C2 protein, a simple chicken IgY purification method was developed, and its purification performance was compared with that of ammonium sulfate precipitation and ethanol fractionation. Quantitative SDS-PAGE analysis showed that the ELP-C2 binding capture method provided a chicken IgY purity of 96.3% and a recovery of 64%, both of which were significantly higher than that of the two traditional methods The ELP-C2 binding capture method could be accomplished within 3 h, in contrast to 30.3 h for ammonium sulfate precipitation or 4.3 h for ethanol fractionation. These data suggest that the ELP-C2 binding capture was a simple, efficient and cost-effective method for purification of chicken IgY. [ABSTRACT FROM AUTHOR]