Recombinant Preparation And Structural Studies Of The Extracellular Domains Of CD81 And Occludin,Cell Surface Receptors For HCV Entry

Hepatitis C virus(HCV)infection is still a very serious global public health problem,and tens of millions of patients with chronic HCV infection are at risk of liver cancer.Although direct-acting antiviral drugs provide a cure for most patients,the high cost of treatment and the risk of re-infection emphasizes the need to develop an potent HCV vaccine that is currently unavailable.The entry process of HCV is a complex multi-step process,but the structural mechanism is not yet clear.HCV envelope glycoproteins(E1 and E2)play an important role in host cell invasion,and research shows that HCV-E2 interacts with host cell receptors CD81,SR-BI,Occludin and Claudin-1.However,except for the CD81 receptor,three-dimensional structures of other HCV receptor proteins(SR-BI,Occludin and Claudin-1)and the complex structures consisting of receptors and HCV envelope proteins have not yet been determined.In this study,crystal structure of the large extracellular loop of CD81(CD81-LEL)with a resolution of 1.8?was determined by X-ray crystallography.The N173-D189 region has multiple conformations,suggesting that this region is relatively flexible.The flexibility may be related to the role of this region in mediating multiple interactions,for example with HCV-E2 and with CD19.Two-dimensional(2D)[¹H-¹⁵N]HSQC spectrum of ¹⁵N-labeled CD81-LEL was analyzed by nuclear magnetic resonance(NMR).In addition,we have compared two methods for preparing¹⁵N-labeled CD81-LEL protein,i.e.,using soluble Thioredoxin-fusion protein and oxidative renaturation from inclusion bodies.The oxidative renaturation method gave the higher yield.These results will help to use the NMR isotopes more cost-efficiently.Furthermore,the extracellular domain of Occludin recombinantly expressed in E.coli was insoluble.The denaturation,purification and oxidative renaturation of the protein was investigated,and results provided a basis for future study on its structure and function.