Recombinant rabbit tissue factor
AssigneePel-Freez
InventorsRobert E. Novy, Jr., Michael J. Domanico, Keith W. Yaeger, Warren Kroeker
Life of this patent
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Abstract
Recombinant rabbit tissue factor is cloned and produced in a bacterial host. This protein, which is relatively insoluble and has several disulfide bonds, requires special modifications in order to express and be purified at commercial levels. By expressing the tissue factor as a fusion protein with a bacterial enzyme, thioredoxin, solubility is increased. Use of a thioredoxin reductase deficient host aids in proper tertiary structure for biological activity.
Claims11
A substantially purified preparation of a fusion protein, the fusion protein comprising, in order from amino terminus toward carboxy terminus:
- the thioredoxin A protein from E. coli;
- multiple contiguous histidine residues;
- a S-peptide;
- and
- a truncated rabbit tissue factor sequence including the extracellular and transmembrane domains, and not the cytoplasmic domain of a native mature rabbit tissue factor protein, wherein the truncated tissue factor protein functions as a part of the blood coagulation cascade.
A method of making recombinant rabbit tissue factor comprising the steps of:
- (a) culturing a thioredoxin reductase deficient bacterial host which harbors a fusion protein coding sequence including a thioredoxin A protein coding sequence, a sequence encoding multiple contiguous histidine residues;
- a S-peptide coding sequence, and a truncated rabbit tissue factor coding sequence including the extracellular and transmembrane domains but not the cytoplasmic domain of the native mature protein, the coding sequences all in a single open reading frame under the control of an inducible promoter, the culturing performed until a desired level of the host is achieved without inducing the promoter;
- (b) cooling the bacterial host to slow its metabolic activity;
- (c) inducing the promoter to initiate fusion protein expressing in the host;
- (d) lysing the host cells;
- and
- (e) recovering the fusion protein from the lysed cells by passage through a column having a specific affinity for one of the multiple histidines or the S-peptide.
A method of making soluble biologically active recombinant rabbit tissue factor comprising the steps of:
- (a) constructing a gene construct encoding a fusion protein including a thioredoxin A protein coding sequence linked to the extracellular and transmembrane domains, but not the cytoplasmic domain, of rabbit tissue factor, the gene construct being under the control of an inducible promoter;
- (b) placing the gene construct in a bacterial thioredoxin reductase deficient host strain deficient in thioredoxin reductase activity;
- (c) culturing the bacterial host strain;
- (d) inducing the promoter in the gene construct to express the fusion protein;
- and
- (e) recovering the fusion protein from the host cells.
A product of manufacture comprising an aqueous solution containing a fusion protein constituting in excess of 90% purity of soluble protein in the solution, the fusion protein having a molecular weight in excess of 40 kDa and having the biological activity, as measured by clotting assay, of rabbit tissue factor, the fusion protein comprising, in order from amino terminus toward carboxy terminus, the thioredoxin A protein from E. coli;
- multiple successive histidine residues;
- a S-peptide;
- and a truncated rabbit tissue factor sequence including the extracellular and transmembrane domains, and not the cytoplasmic domain of the native mature protein.
A substantially purified preparation of a fusion protein, the fusion protein comprising, in order from amino terminus toward carboxy terminus;
- the thioredoxin A protein from E. coli;
- at least six contiguous histidine residues;
- a S-peptide;
- and
- a truncated rabbit tissue factor sequence including the extracellular and transmembrane domains, and not the cytoplasmic domain of the native mature protein, the truncated tissue factor protein being capable of functioning as a part of the blood coagulation cascade;
- wherein the fusion protein has the sequence of SEQ ID NO:2.
Dependencies are inferred from the claim text.