Preparation And PEGylation Of Recombinant Human Interferon Lambda3

Objective:Human interferon lambdas (hIFN-λs) have recently been identified as new types of interferons. It is now clear that IFN-λs are important mediators of antiviral responses in mucosal/epithelial tissues, and are critically important for the protection of GI epithelium. Nevertheless, important aspects of IFN-λs biology require further experimental exploration to advance our understanding of the complex role of type III IFNs in overall immunity. We chiefly studied IFN-λ3and wanted to express it in E. coli, and then prepare PEGylated recombinant human interferon lambda3(PEG-rhIFN-λ3). Furthermore, biological activity of the purified rhIFN-λ3and the mono-pegylated rhIFN-X3was preliminary evaluated.Methods:The rhIFN-X3gene was synthesized and inserted into pThioHisA vector after codon optimization and transformed into E. coli Top10strain, and then it was induced with IPTG. Then the expressed protein was refolded by dialysis, purified with ion-exchange chromatographic. Following the recombinant protein was subjected to mPEG-ButyrALD (Mr:10kDa) modification after EK cleavage digestion and chromatographic purification. Subsequently, the modified product was preliminary isolated and purified for determining its activity.Results:The recombinant plasmid pThioHisA-rhIFN-λ3was successfully constructed as verified by restriction endonucleases digestion, PCR amplification and following DNA sequencing.The recombinant protein was expressed in the form of inclusion bodies after induced by1mmol/L IPTG. After dialysis, renaturation, EK cleavage digestion, and ion exchange chromatography purification, a soluble rhIFN-λ3protein was obtained with a molecule of19kDa which was verified by western blot analysis. The purity of the purified rhIFN-λ3was as high as90%. The final condition for PEGylation was rhIFN-λ3reacted with10-fold molar excess of the10kDa mPEG2-butyrALD in50mM phosphate,5mmol/L sodium cyanoborohydride, pH6.5, at room temperature for8hours. The mono-PEGylated rhIFN-λ3with a purity of86%was successfully obtained after cation-exchange chromatography. The50%effective concentration (EC50) of rhIFN-λ3in WISH cells against VSV was8.43ng/mL, while the EC50of mono-PEGylated rhIFN-λ3was49.19ng/mL, which reserved17.14%of the in vitro activity.Conclusion:In present study, rhIFN-3was successfully expressed, purified and modified. Both of the purified rhIFN-λ3and the mono-pegylated rhIFN-λ3showed potency against antiviral activity in vitro and supported further studies of this new type of investigational interferon. Further study is needed to better understand the in vivo immunogenicity, antigenicity, stability and antiviral activity of PEG-rhIFN-λ3.