Identification of porcine coagulation factor VIII domains responsible for high level expression via enhanced secretion

J Biol Chem. 2004 Feb 20;279(8):6546-52. doi: 10.1074/jbc.M312451200. Epub 2003 Dec 1.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • COS Cells
  • Cricetinae
  • DNA, Complementary / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Factor VIII / chemistry*
  • Factor VIII / metabolism*
  • Humans
  • Mice
  • Precipitin Tests
  • Protein Structure, Tertiary
  • RNA / chemistry
  • RNA, Messenger / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Swine
  • Transgenes

Substances

  • DNA, Complementary
  • RNA, Messenger
  • Recombinant Fusion Proteins
  • RNA
  • Factor VIII