Prokaryotic And Eukaryotic Expression Of Von Willebrand Factor A1A2A3 Triplet And Its Biological Activities

von Willebrand factor (vWF) is a multimeric adhesive protein that is synthesized in endothelial cells and megakaryocytes/platelets. This huge protein with a unique multimeric structure plays a pivotal role in hemostasis and thrombosis.von Willebrand disease will development once there is quality or quantity of vWF.In this process vWF promotes platelet adhesion to the subendothelium of damaged blood vessels and blood coagulation through its multiple interactions with the platelet membrane receptors,glycoprotein (GP)Ⅰb-Ⅸ-Ⅴcomplex,GPⅡb-Ⅲa complex,and exposed endothelial various types of collagen.Platelet adhesion is the initial event in thrombosis.Whereas ultra-large (UL) and highly adhesive forms of vWF multimers potentially causing systemic or localized thromboembolism as found in thrombotic thrombocytopenia purpura.The theme of this study is to focus on the functions of different domains of vWF according to the relation of collagen-vWF-GPIb,using the technology of gene recombination to clone the gene fragment of vWF A1A2A3 function structural domain from human total length vWF cDNA.We express vWF A1A2A3 domain in E.coli and CHO cell,and compare the biological function of the two recombinate proteinum.Objective:The A1 domain contains binding sites for platelet glycoprotein(GP)Ⅰba,ristocetin,heparin,sulfatides and collagen. Its homologous A3 domain only binds to collagen fibrils typesⅠandⅢ, whereas the A2 domain contains the cleavage site for the metalloprotease ADAMTS-13(a disintegrin-like and metallo-protease with thrombospondin type 1 motif).To study the biological activity of vWF-AlA2A3,we consbuct prokaryotic and eukaryotic expression vectors harboring homo sapiens vWF-A1A2A3 gene,and express vWF A1A2A3 domain in E.coli and CHO cell.Methods:The human vWF-A1A2A3 cDNA was amplied from the human vWF cDNA by PCR and the PCR products which was in line with the reported human vWF-A1A2A3 gene were inserted into prokaryotic vectors PET-21b and eukaryotic expression vectors pSectag2b-A1A2A3 by enzyme restriction and ligation.The recombinant expression plasmid was transfected E.coli into CHO cells and the transient.After using Ni-NTA agarose column to purify expression product.which was analyzed by Western blotting.Enzyme linked immunosorbent assay(ELISA) was used to study biological function of recombination protein through collagen binding test and platelet binding under ristocetin induction.Undering urea degenerating,ADAMTS-13 hydrolyses recombination protein,then use western blotting identification.Results:We adopted reverse transcription-PCR to amplify cDNA of vWF A1A2A3 domain from human total length vWF cDNA. After sequence analysis, the amplified DNA fragments were inserted into expression vectors with 6×his taq (pET-21b, pSectag2b). The recombinant expression vectors were transformed into E. coli, and CHO.The recombinant protein expressed in E. coli was purified by chromatography on Ni-NTA agarose column and renatured by Tris.HCl buffer containing GSH and GSSG, their purity were above 95%. The resulting protein was refolded by dialysis.Then we do some research about its biological function. Eukaryotic expression using serum-free culture has the superiority that need't process denaturation and renaturation, and reserve the spatial structure of recombinant protein by largest extent.Experimental result reveales that recombinant vWF-A1A2A3(rvWF-A1A2A3) has correct immunology and biologic activity.rvWF-A1A2A3 can bind specificity with anti-his antibody,SZ-34,SZ-123,SZ-125 and SZ-129 antibody,western blot shows respectively single strap.It was identified to have the abilities to bind humanⅢcollagen and platelet under ristocetin-induced.The recombinant protein expressed in CHO has better affinity than in E.coli,and it has significant statistics disparity, S=10.5,P=0.0313,p<0.05.rvWF-A1A2A3 protein expressed in eukaryotic cell under containning urea,Ca2+/Zn2+ buffer can be cutted by ADAMTS-13.Conclusion:rvWF-AlA2A3 was successfully expressed in E.coli and CHO.The recombinant protein has fine immunology and biological function activity.rvWF-AlA2A3 protein provides a basis for the further study on its biological structure, function and clinical application...