Process for preparing functional recombinant tissue factor

Details Process for preparing functional recombinant tissue factor Process for preparing functional recombinant tissue factor

1998-11-06

2001-07-17

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S071200, C530S350000, C530S381000, C530S412000, C530S413000, C530S416000, C530S418000, C530S421000

Reexamination Certificate

active

06261803

ABSTRACT:

The present invention relates to a process for preparing functional recombinant tissue factor in a prokaryotic host organism.
The contact of blood with tissue surfaces leads to a marked acceleration in coagulation. This acceleration is due to the specific effect of a factor from the tissue. This “tissue factor” is a protein which has to be associated with phospholipids in order to be able to act in a procoagulatory manner. The protein was purified for the first time from bovine brain in 1980 (Bach et al., J. Biol. Chem. 256 (16), 8324 (1981)); human tissue factor was also subsequently purified (Broze et al., J. Biol. Chem. 260 (20), 10917 (1985)). Amino acid sequences of this protein were used to design oligonucleotide probes which enabled the protein to be cloned. The primary translation product is a polypeptide composed of 295 amino acids. The surface domain, together with the receptor domain, takes up the 219 N-terminal amino acids. A transmembrane domain of 23 amino acids in length and a cytoplasmic moiety of 20 amino acids in length follow in the C-terminal direction (Edgington et al., Thromb. Haemostas. 66 (1), 67 (1991)). The solitary cysteine in the cytoplasmic domain can serve as the acceptor for a palmitate or stearate residue, which is linked by way of a thioester bond. Two intramolecular disulfide bridges are located in the extracellular domain; the C-terminal of these is required for binding factor VIIa. The surface domain is glycosylated by way of three threonine residues.
According to the currently accepted model of the initiation of coagulation, a blood vessel lesion, for example, leads to blood making contact with endothelial cells. Factor VII or factor VIIa from blood plasma binds to the tissue factor receptor on the endothelial cells. In the presence of calcium and phospholipid, factor X is converted into factor Xa by the complex on the cell surface. The factor Xa in turn converts prothrombin into thrombin, while the latter converts fibrinogen into fibrin. Finally, a local clot is formed.
The protein has been expressed in various systems using the cloned human cDNA. For example, Paborsky et al., Biochemistry 28, 8072 (1989), report its overexpression in
E. coli.
The expression of eukaryotic proteins in
E. coli
is associated with a number of fundamental problems. One of these basic problems is that the bacteria lack their own glycosylation systems. Proteins whose functional activity depends on a glycosylation cannot be expressed in active form in
E. coli
. Another problem is the high activity of cell-specific proteases in
E. coli
(Maurizi et al., Experientia 48, 176 (1992)), which activity limits the stability of the translation products which are formed from inserted foreign genes. Finally, a fundamental problem associated with expressing tissue factor in
E. coli
is that this protein is a membrane protein. It is known that the rate at which membrane proteins are expressed is markedly lower than that at which soluble proteins are expressed, presumably because the uptake capacity of the cellular compartment in which the over-expressed molecules become deposited, i.e. the membranes of the bacterial cells, is limited. While it has already been reported that this uptake capacity can be increased by forming lamellar membrane structures (intracellular membrane invaginations), which are similar to those in mitochondria or chloroplasts, these structures have so far only been observed when
E. coli
-specific membrane proteins are being overexpressed and not in the case of heterologous eukaryotic proteins (“exclusion bodies”, von
Meyenburg, K. et al. EMBO Journal, Vol.
3, 1791--1797, 1984
).
An alternative strategy is therefore to express a mutated tissue factor molecule which lacks the transmembrane domain. This so-called “soluble” tissue factor accumulates in the cytoplasm of the bacterial cells and can be expressed in
E. coli
in relatively large quantities. However, in this system, the problem can arise that the tissue factor is present in the
E. coli
cell in a quasicrystalline state in the form of so-called inclusion bodies. When this is the case, the inclusion bodies have to be solubilized by using very large quantities of chaotropic agents, and the proteins which have been monomerized in this way have then once again to be refolded, with a great deal of effort and usually with only a low yield, into an active, renatured confirmation.
However, in principle, the soluble tissue factor is not suitable for use in prothrombin time reagents since it lacks the domain for the interaction with phospholipids. It would only be possible to avoid these difficulties by integrating the native protein into a lipid membrane during biosynthesis of the protein. The particular advantage of the procedure would be that, on the one hand, the stability of the protein would be increased as a result of integration into the membrane and, on the other hand, this integration would also stabilize the biologically active confirmation. Another approach to overexpressing the tissue factor is that of using large number of known and successfully employed expression systems which encode products of gene fusions (e.g. with &bgr;-galactosidase, MalE, glutathione transferase and His-tag). However, these systems are not suitable for expressing biologically active tissue factor. While expression products can indeed be detected, and the level of expression can also be increased, when these systems are used, this is at the same time associated with complete loss of function, which cannot be restored, either, even using elaborate renaturation methods.
The various problems of overexpression in
E. coli
which have been mentioned can be circumvented by carrying out the expression in a eukaryotic system. Thus, expression in yeast, in an insect cell culture using baculovirus as a vector, or in cultured mammalian cells, for example hamster ovary cells, or in human cell lines (Paborsky et al., 1989), for example, is in principle suitable. However, these systems suffer from crucial disadvantages with regard to cost.
There is so far no known process for preparing large quantities of complete, biologically active, recombinant tissue factor from
E. coli
in high yield and at a high level of purity.
Strategies for purifying native and recombinant tissue factor are described in the literature, e.g. in Paborsky et al., 1989. As a rule, a detergent, for example deoxycholate or Triton-X 100, is used to extract the tissue factor from cells or tissue. The purification strategies contain a variety of chromatography processes. These extend from gel filtration via ion exchange chromatography and hydrophobic interaction chromatography through to steps involving affinity chromatography. Factor and antibodies against tissue factor are described as affinity materials (Paborsky et al., 1989). A feature common to the published purification methods is that their use is restricted to a fermenter volume of up to 1 L. There has previously been no description of a robust method for preparing relatively large quantities of pure tissue factor on a pilot plant scale (fermenter volume of 10-100 L).
Tissue factor is used in prothrombin time reagents, for example. In this case, a blood plasma sample is coagulated with an excess of tissue factor in the presence of phospholipids and calcium. That which is diagnostically relevant is the coagulation time, which can be converted, for example, into activity values in % of the standard, or into prothrombin ratio values, using suitable calibration systems. The test is used for screening the extrinsic coagulation system, for example before operations, for checking the activity of the individual factors of the extrinsic coagulation system, and for monitoring therapy in association with oral anticoagulant therapy.
Other diagnostic uses of the tissue factor are also conceivable in addition to this standard use, for example in a test for the tissue factor pathway inhibitor, in neutralization tests for lupus anticoagulants or as an amplification and detection system for immunological tests.

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