Purification of recombinant human tissue factor

Abstract

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.

Comparison of natural and recombinant tissue factor proteins: new insights

“…The N-terminal analysis showed that both recombinant and placental proteins exist in two forms, a full-length form composed of 263 amino acid residues and a truncated form composed of 261 residues, where the first two amino acids on the N-terminus of the protein are missing. This observation was consistent with previously published data (Spicer et al , 1987 ;Paborsky et al , 1989 ). The fractional abundance of each form in the two proteins is different.…”

“…The amino acid sequence data indicate that full-length TF has three potential glycosylation sites at Asn ) in the cytoplasmic domain (Paborsky et al , 1989 ;Paborsky and Harris , 1990 ). The latter site is not present in truncated TF proteins.…”

“…A variety of human recombinant TF species have been produced, from those containing only the extracellular domain to the full-length protein (Paborsky et al , 1989 ;Fiore et al , 1994 ). These recombinant proteins have been extensively used in research and clinical laboratories worldwide because of the limited availability of natural TF.…”

Endothelial cells, tissue factor and infectious diseases

“…Tissue factor triggers the extrinsic coagulation pathway, plays a key role in homeostasis and in several thrombotic diseases, and is also involved in cell signaling (4)(5)(6)(7)(8)(9)(10). Tissue factor is a glycosylated membrane protein that consists of a single polypeptide chain of 263 amino acids with an apparent molecular mass of 46 kDa, as determined by SDS-PAGE electrophoresis (5,(11)(12)(13)(14). Glycosylation does not appear to be important for tissue factor function as the recombinant nonglycosylated protein retains procoagulant activity (12).…”

“…Tissue factor is a glycosylated membrane protein that consists of a single polypeptide chain of 263 amino acids with an apparent molecular mass of 46 kDa, as determined by SDS-PAGE electrophoresis (5,(11)(12)(13)(14). Glycosylation does not appear to be important for tissue factor function as the recombinant nonglycosylated protein retains procoagulant activity (12). Tissue factor is a type I integral membrane protein.…”