Purification of recombinant human tissue factor.
Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.
Paborsky
L R
LR
Department of Cardiovascular Research, Genentech, Inc., South San Francisco, California 94080.
Tate
K M
KM
Harris
R J
RJ
Yansura
D G
DG
Band
L
L
McCray
G
G
Gorman
C M
CM
O'Brien
D P
DP
Chang
J Y
JY
Swartz
J R
JR
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003488:Cyanogen Bromide; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D011517:Prothrombin Time; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013925:Thromboplastin / Q000302:isolation & purification* / Q000494:pharmacology; D014357:Trypsin
D011994:Recombinant Proteins; D013925:Thromboplastin; D014357:Trypsin; D003545:Cysteine; D003488:Cyanogen Bromide