Expression and purification of recombinant rabbit factor VII.
To facilitate studies of the in vivo role of the extrinsic pathway of coagulation in experimental hemostasis and thrombosis, a full-length cDNA-encoding rabbit factor VII was isolated using polymerase chain reaction-mediated DNA amplification from plaque-purified lambda gt11 phage. Repeated DNA sequencing of both full-length rabbit factor VII cDNA and shorter cDNA fragments verified four changes in the previously reported amino acid sequence of mature rabbit factor VII, now predicted to be 405 amino acids in length. Rabbit factor VII cDNA was transfected into human embryonic kidney 293 cells and a cell line that permanently expressed rabbit recombinant factor VII was established. Rabbit recombinant factor VII was purified from tissue culture 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 authenticity of rabbit recombinant factor VII was confirmed by polyacrylamide gel electrophoresis and Western blot analysis. Homogeneous rabbit recombinant factor VII was fully active biologically as determined by prothrombin time assay 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 coagulopathies.
Ruiz
S M
SM
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada.
Sridhara
S
S
Blajchman
M A
MA
Clarke
B J
BJ
D000818:Animals; D000906:Antibodies; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005167:Factor VII / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D005167:Factor VII