The Expression Purification And Behavioral Characterization Of NTCP

Sodium-taurocholate cotransporting polypeptide(NTCP)is a member of the tenth solute carrier(SLC10)family.The NTCP protein specifically expressed in the basement membrane of liver cells that are responsible for the regulation of bile acid enterohepatic circulation of bile acids in order to maintain the balance of the body.The latest study found that the NTCP protein is functional receptors for hepatocytes infected by Hepatitis B virus and Hepatitis D virus.Therefore,obtaining the structure of NTCP is of great significance for the treatment of hepatitis B and further elucidation of the functions of NTCP.In this study,the NTCP proteins of human,mouse,rat,tree shrew,and cynomolgus monkeys were used as research objects.Based on the NTCP fragments of these five species,corresponding primers were designed to clone the five NTCP gene fragments.The recombinant expression vector was constructed and insect cells were used as the expression host to obtain the target protein using the Bac-to-Bac baculovirus expression system.The target protein was enriched and roughly extracted by Ni column affinity chromatography;then the AKTA FPLC purification system was used to observe the binding of the target protein with different detergents through Superdex200;and the right detergent was find to Purify protein and prepare for crystallization.In this study,the NTCP gene fragments of human,mouse,rat,tree shrew,and cynomolgus monkeys were obtained by PCR.These NTCP gene fragments and p Fast Bac with different tags were used to construct a recombinant vector.The DH10 competent cells were transformed with these recombinant vectors;and NTCP recombinant Bacmid with different labels were obtained through screening with blue and white spots.The NTCP recombinant Bacmid were transfected into insect cells with a transfection reagent to obtain recombinant baculoviruses for infecting insect cells;and these NTCP proteins with different tags were expressed in insect cells.By comparing the expression levels of these NTCP proteins in insect cells,it was found that the expression of NTCP protein of tree shrew with SUMO tag or MBP tag was higher.Observing the binding of NTCP protein with MBP tagged tree ticks to Superdex200 and different detergents,it was found that the NTCP protein of tree shrew has better behavior in detergent C325 S.Finally,when purifying the NTCP protein of the tree shrew,the NTCP protein of the tree shrew was combined with the polypeptide Myr47,and the behavior of the protein was observed in the detergent C325 S.It was found that after the target NTCP protein was combined with the Myr47 polypeptide,the NTCP protein of the polymerized tree shrew was reduced.The research of this topic provides some reference value for the research of anti-HBV drugs.