title: Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion.
abstract: Blood coagulation factor VIII has a domain structure designated A1-A2-B-ap-A3-C1-C2. Human factor VIII is present at low concentration in normal plasma and, comparably, is produced at low levels in vitro and in vivo using transgenic expression techniques. Heterologous expression of B domain-deleted porcine factor VIII in mammalian cell culture is significantly greater than B domain-deleted human or murine factor VIII. Novel hybrid human/porcine factor VIII molecules were constructed to identify porcine factor VIII domains that confer high level expression. Hybrid human/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domains expressed at levels comparable with recombinant porcine factor VIII. A hybrid construct containing only the porcine A1 domain expressed at intermediate levels between human and porcine factor VIII, whereas a hybrid construct containing the porcine ap-A3 domain expressed at levels comparable with human factor VIII. Additionally, hybrid murine/porcine factor VIII constructs containing the porcine factor VIII A1 and ap-A3 domain sequences expressed at levels significantly higher than recombinant murine factor VIII. Therefore, the porcine A1 and ap-A3 domains are necessary and sufficient for the high level expression associated with porcine factor VIII. Metabolic radiolabeling experiments demonstrated that high level expression was attributable to enhanced secretory efficiency.
authors.affiliation: Winship Cancer Institute, Emory University, Atlanta, Georgia 30322, USA.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Doering
authors.forename: John F
authors.initials: JF
authors.lastname: Healey
authors.forename: Ernest T
authors.initials: ET
authors.lastname: Parker
authors.forename: Rachel T
authors.initials: RT
authors.lastname: Barrow
authors.forename: Pete
authors.initials: P
authors.lastname: Lollar
mesh_terms: D000818:Animals; D019556:COS Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D012313:RNA / Q000737:chemistry; D012333:RNA, Messenger / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013552:Swine; D019076:Transgenes
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012313:RNA; D005169:Factor VIII