Cloning and expression in COS-1 cells of a full-length cDNA encoding human coagulation factor X.
A 1.5-kb cDNA (FX) encoding full-length human coagulation factor X was isolated from a human fetal liver cDNA library. The identity of the insert in a selected phage lambda clone was confirmed to be FX by nucleotide (nt) sequence analysis and restriction mapping. This FX cDNA clone contained 1467 bp of coding sequence, no 5'-untranslated sequence, a short 3'-untranslated sequence of 10 nt and a poly(A) tail at the 3'-end. The FX cDNA was inserted into a mammalian expression vector and transfected into COS-1 monkey kidney cells. Media from transfected cells showed evidence of factor X antigen and, following addition of Russel's viper venom factor X activator, enhanced amidolytic activity toward a synthetic peptide rho-nitroanilide substrate. Immunoprecipitation with an anti-factor X monoclonal antibody of [35S]methionine-labeled cell-conditioned media showed evidence of polypeptides of 74, 55, and 17 kDa, as determined by SDS-PAGE followed by autoradiography. Together, these results indicate that an active factor X can be successfully expressed in a recombinant DNA expression system. This approach will allow the systematic structure/function investigation of this important blood-clotting enzyme.
Messier
T L
TL
Department of Biochemistry, University of Vermont, Burlington 05405.
Pittman
D D
DD
Long
G L
GL
Kaufman
R J
RJ
Church
W R
WR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000032:analysis*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005170:Factor X / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression / Q000187:drug effects; D000882:Haplorhini / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D014162:Transfection; D014812:Vitamin K / Q000494:pharmacology
D004274:DNA, Recombinant; D014812:Vitamin K; D005170:Factor X