Human coagulation factor VIII domain-specific recombinant polypeptide expression
نویسندگان
چکیده
BACKGROUND Hemophilia A is caused by heterogeneous mutations in F8. Coagulation factor VIII (FVIII), the product of F8, is composed of multiple domains designated A1-A2-B-A3-C1-C2. FVIII is known to interact with diverse proteins, and this characteristic may be important for hemostasis. However, little is known about domain-specific functions or their specific binding partners. METHODS To determine F8 domain-specific functions during blood coagulation, the FVIII domains A1, A2, A3, and C were cloned from Hep3B hepatocytes. Domain-specific recombinant polypeptides were glutathione S-transferase (GST)- or polyhistidine (His)-tagged, over-expressed in bacteria, and purified by specific affinity chromatography. RESULTS Recombinant polypeptides of predicted sizes were obtained. The GST-tagged A2 polypeptide interacted with coagulation factor IX, which is known to bind the A2 domain of activated FVIII. CONCLUSION Recombinant, domain-specific polypeptides are useful tools to study the domain-specific functions of FVIII during the coagulation process, and they may be used for production of domain-specific antibodies.
منابع مشابه
Expression of Biologically Active Recombinant B-Domain-Deleted Human Factor VIII in Mammalian Cells
متن کامل
The Effects of Novel Mutations in A1 Domain of Human Coagulation Factor VIII on Its Secretion Level in Cultured Mammalian Cells
Inefficient secretion of the human coagulation factor (hFVIII) in mammalian expression systems is one ofthe main causes of the hFVIII low expression level, attributed to its interaction with a chaperone known asBiP/GRP78. In order to improve secretion efficiency of the hFVIII, based on the higher secretion level of theporcine FVIII and analysis of the hFVIII A110 region, that ...
متن کاملA2 domain of human recombinant-derived factor VIII is required for procoagulant activity but not for thrombin cleavage.
Thrombin treatment of the coagulation factor VIII results in a rapid activation of procoagulant activity with a subsequent first order decay. The structural requirements for thrombin-activated factor VIII were characterized using recombinant-derived human factor VIII and site-directed DNA-mediated mutagenesis. Thrombin-activated human recombinant-derived factor VIII was isolated in an active fo...
متن کاملExpression of Recombinant Coagulation Factor IX in Human Amniotic Membrane-derived Mesenchymal Stem Cells: A New Strategy to Gene Therapy of Hemophilia B
Background: Hemophilia B is an X-linked hereditary disorder of blood coagulation system which is caused by factor IX (FIX) deficiency. Factor IX is a plasma glycoprotein that participates in the coagulation process leading to the generation of fibrin. Replacement of factor IX with plasma-derived or recombinant factor IX is the conventional treatment for hemophilia B to raise the factor IX le...
متن کاملBacterial Expression and Purification of C1C2 Domain of Human Factor VIII
With the aim of the production of human factor VIII antigen and its corresponding antibody an epitope coding fragment of the light-chain of hFVIII, fused to a His6-tag, was isolated and over-expressed in Escherichia coli. The over-expressed hFVIII-epitope containing peptide was confirmed by its reaction with a rabbit serum directed against native hFVIII as well as antiHis6-tag antibody. An expr...
متن کامل