Expression and purification of recombinant rabbit factor VII

To facilitate studies of the in vivo role of the extrinsic pathway of coagu lation in experimental hemostasis and thrombosis, a full-length cDNA-encodi ng rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage, Repeated DNA sequ encing of both full-length rabbit factor VII cDNA and shorter cDNA fragment s verified four changes in the previously reported amino acid sequence of m ature rabbit factor VII, now predicted to be 405 amino acids in length. Rab bit factor VII cDNA was transfected into human embryonic kidney 293 cells a nd a cell line that permanently expressed rabbit recombinant factor VII was established. Rabbit recombinant factor VII was purified from tissue cultur e media using a combination of barium citrate precipitation, DEAE-sepharose FF chromatography, benzamidine agarose, and affinity chromatography using a sheep antirabbit factor VII polyclonal antibody, The purity and authentic ity of rabbit recombinant factor VII was confirmed by polyacrylamide gel el ectrophoresis and Western blot analysis. Homogeneous rabbit recombinant fac tor VII was fully active biologically as determined by prothrombin time ass ay in factor FVII-depleted plasmas, of both human and rabbit origin, using either human or rabbit thromboplastin, Rabbit recombinant factor VII should prove useful for future in vivo investigations of experimental coagulopath ies. (C) 2000 Elsevier Science Ltd. All rights reserved.