Structural Studies Of Novaferon And Interferon Receptor Complex

Interferon(IFN)is a class of cytokines with antiviral,antiproliferative and antitumor activities,and also shows a regulatory effect on innate and adaptive immune responses.Interferons are produced in response to viral and microbial infections,their most outstanding feature is the ability to nonspecifically inhibit viral growth by inducing the�antiviral state�in cells.The human interferons are divided into three types,based on amino acid sequence homology and the receptors.Type I interferons include IFN ?,?,?,?,?,?,? and ?,while type II and type III interferon have only one member,IFN ? and IFN ?,respectively.Interferons are produced naturally in small quantities.The emergence of DNA recombination technology makes large-scale production of interferon possible,which also promotes the clinical research of interferon.At present,most of the interferons used in clinic belong to type I interferon family.IFN a is currently the most used cytokine in the treatment of cancerNovaferon(Nova)is a novel recombinant IFN a anti-tumor/viral protein.Screened from over 100 thousand artificial clones of recombinant interferons by DNA shuffling and high-throughput screening techniques.Previous literature and clinical studies have shown that Novaferon has stronger bioactivity than natural IFN a,and have the same mechanism on cancer treatment in cell cycle arrest,promotion of apoptosis and inhibition of oncogene expressionIn this paper,the Novaferon protein was expressed and purified,and its biological activity was determined by cytopathic inhibition.By screening and optimizing the crystallization conditions of the protein,we obtained crystals with better resolution and resolved the structure.Next,the extracellular domain of interferon receptor IFNAR2 was expressed by Drosophila S2 cells,and the protein with higher purity was obtained through affinity chromatography and gel filtration chromatography.The interaction between Novaferon and IFNAR2 was verified by isothermal titration calorimetry(ITC)and biofilm interferometry(BLI).We obtained Novaferon and IFNAR2 complex protein crystals by screening the crystallization conditions,but the crystal resolution was poor.Later,we tried to optimize the crystals by deglycosylation and in situ protein hydrolysis.We hope to obtain crystals with higher resolution and resolve the structure of the complex,laying a foundation for revealing the mechanism of Novaferon high biological activity.