RU2321633C2 - Method for preparing vitamin k-dependent proteins - Google Patents

Abstract

FIELD: biotechnology, biochemistry, molecular biology.

SUBSTANCE: invention represents eucaryotic cell-host that expresses the first polynucleotide encoding the first propeptide and FVII or its functional variants and relates to the first expression unit, and expresses the second polynucleotide encoding the second free propeptide. Each of indicated the first and second propeptides comprises amino acid sequence chosen independently from group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. Also, invention relates to a method for preparing vitamin K-dependent proteins, among them and FVII, using abovementioned cell-host, and to a method for preparing this cell-host. Invention provides preparing vitamin K-dependent proteins with the high degree of effectiveness.

EFFECT: improved preparing method of proteins.

38 cl, 3 tbl, 3 dwg, 2 ex

Description

The present invention relates to a new method for producing vitamin K-dependent proteins and, in particular, coagulation factor VII (FVII). In addition, the present invention relates to new cotransfected eukaryotic host cells and recombinant vectors to be used in this improved method for producing vitamin K-dependent proteins.

BACKGROUND OF THE INVENTION

The biosynthesis of vitamin K-dependent proteins involves several stages of post-translational processing before a mature functional protein is obtained.

Vitamin K is a necessary cofactor for gamma-carboxylation of glutamic acid residues in these vitamin K-dependent proteins, including procoagulant factors: thrombin, factor VII, IX and X; anticoagulants, protein C and protein S and other proteins such as osteocalcin (bone protein Gla), matrix protein Gla and proline-enriched protein Gla 1. This carboxylation is necessary for normal hemostasis, as it allows calcium binding and the attachment of procoagulants and anticoagulants to phospholipids.

Gamma-glutamyl carboxylase is an integrated membrane microsomal enzyme located in the crude endoplasmic reticulum. It carboxylates the glutamate residues located in the Gla domain of vitamin K-dependent proteins. Human gamma glutamyl carboxylase cDNA has recently been isolated and sequenced (Wu SM et al. Science 254: 1634, 1991). Studies of the biological synthesis of vitamin K-dependent proteins in BHK and CHO cells showed that carboxylase is present both in the endoplasmic reticulum (ER) and in the Golgi complex and that the propeptide containing the carboxylase recognition site is cleaved after gamma-carboxylation is completed.

It was discussed whether a propeptide can stimulate carboxylase activity (Sigiura, I. et al. (1997) Proc. Natl. Acad. Sci., 9, 9069-9074, Knobloch and Suttie (1987) J. Biol. Chem. 262, 15334- 15337, Furie et al. (1999) Blood, 93, 1798-1808).

Blood coagulation is a process consisting of the complex interaction of various blood components or factors that may lead to the formation of a fibrin thrombus. In general, the blood components that take part in this are referred to as the “coagulation system”, are proenzymes or zymogens, enzymatically inactive proteins that are converted to proteolytic enzymes by the activator, the most activated coagulation factor. Coagulation factors that undergo such a transformation are generally referred to as “active factors” and are indicated by adding a subscript “a” (for example, activated factor VII (FVIIa)).

Activated factor X (FXa) is necessary for the conversion of prothrombin to thrombin, which then converts fibrinogen to fibrin, as the final step in the formation of a fibrin thrombus. There are two systems or two paths that contribute to the activation of FX. The "internal pathway" refers to those reactions that lead to the formation of thrombin by using factors present only in plasma. In the end, a series of protease-mediated activation processes generate factor IXa, which, in combination with factor VIIIa, cleaves FX to FXa. A similar proteolysis is carried out using FVIIa and its cofactor, a tissue factor, on the “external pathway” of blood coagulation. Tissue factor is a membrane-bound protein and usually cannot circulate in blood plasma. However, upon vessel destruction, it can complex with FVIIa with catalysis of FX activation or activation of factor IX in the presence of Ca ⁺⁺ and phospholipid. Although the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years, FVII and tissue factor have been found to play a major role in the regulation of blood coagulation.

FVII is a glycoprotein present in small amounts in plasma that circulates in the blood as a single-chain zymogen. Zymogen is not active in relation to coagulation. Single chain FVII can be converted to double chain FVIIa using FXa, factor XIIa, factor IXa or thrombin in vitro. FXa is thought to be the main physiological activator of FVII. Like several other plasma proteins involved in hemostasis, FVII depends on vitamin K during its biosynthesis, which is necessary for gamma-carboxylation of 10 glutamic acid residues in the amino terminus of the protein. The intracellular post-translational treatment of FVII takes place in the endoplasmic reticulum (ER) and in the Golgi complex. Along with vitamin K-dependent gamma-carboxylation, FVII undergoes limited proteolysis to remove the propeptide with the N-terminus and for glycosylation of asparagine-145 and -322 and serine-52 and -60 (Fig. 1).

Residues of gamma-carboxylated glutamic acid (Gla) are necessary for the metal-bonded interaction of FVII with phospholipids.

In the presence of tissue factor, phospholipids, and calcium ions, double-stranded FVIIa rapidly activates FX or factor IX via limited proteolysis.

Protein C is a serine protease and a naturally occurring anticoagulant that plays a role in the regulation of homeostasis by inactivating factors Va and VIIIa in the blood coagulation system. In vivo human protein C is produced primarily in the liver as a single polypeptide of 461 amino acids. This single chain precursor molecule undergoes many post-translational modifications, including carboxylation of nine glutamic acid residues, resulting in nine Gla residues.

Protein S also exhibits anticoagulant activity in in vitro coagulation assays. Protein S shows anticoagulant cofactor activity for activated protein C. Also, protein S is shown to be an anticoagulant factor in the absence of activated protein C, since it can inhibit prothrombinase activity in assays that do not use activated protein C and binds to factor Va or factor Xa and functions as an anticoagulant without activated protein C.

Protein S is a physiologically very important antithrombotic factor, since hereditary or acquired protein S deficiencies are associated with venous and arterial thrombotic disease. Deficiency of free S protein at a normal level of S protein is generally described in some patients with thrombotic disease.

It is often necessary to selectively block the patient’s blood coagulation system. Anticoagulants such as protein C or protein S can be used, for example, during renal dialysis or for the treatment of deep vein thrombosis, disseminated intravascular coagulation (DIG), a patient at risk of acute thrombosis, protein S deficiency, sepsis, inflammation, and cancer undergoing surgery, and owners of other medical disorders.

Osteocalcin consists of 49 amino acid residues, which include three Gla residues. The function of this protein is supposed to be to suppress excess mineralization. Osteocalcin is a specific bone protein that is secreted by osteoblasts. The fraction of newly synthesized osteocalcin is released into the blood stream, where its concentration correlates with indicators of osteoblast activity and bone formation rate. In humans, changes in circulating osteocalcin levels are associated with metabolic bone diseases such as osteoporosis and hyperparathyroidism.

Gla matrix protein (MGP) consists of 79 amino acids, including 5 Gla residues. This protein is usually found in the demineralized matrix and is supposed to perform a specific function when initiating bone formation.

There remains a need in the art for improved systems for producing recombinant vitamin K-dependent proteins and specific recombinant coagulation factors. The present invention satisfies this need by providing a method that provides more efficient, faster production and / or higher yield of recombinant vitamin K-dependent proteins, in particular FVII.

Description of the invention

The present invention relates to a new method for producing vitamin K-dependent proteins and, in particular, coagulation factor VII (FVII).

The processing steps of the N-termini and, in particular, the gamma-carboxylation of glutamic acid residues of vitamin K-dependent proteins, as shown, are the limiting stages in the biosynthesis of these proteins, as shown by the authors of the present invention regarding FVII. A method for increasing gamma-carboxylation, as shown, increases the expression of recombinant vitamin K-dependent proteins.

The propeptide, vitamin K-dependent proteins, is most important for binding to carboxylase and it must be covalently attached to the vitamin K-dependent protein in the same order as for Glu residues, where the N-terminus of the future mature protein will be carboxylated. The authors suggest that the free propeptide functions as a molecule for allosteric regulation, in the sense that when the concentration of the free propeptide increases, the activity of the gamma-carboxylation process also increases.

A direct increase in the concentration of free propeptides can be obtained by co-expression of the free propeptide (free propeptides) per se. This can be done by transfecting with a polynucleotide encoding a free propeptide (free propeptides), with or without mutations and post-translational modifications, in one or more copies, and co-expressing it together with a polynucleotide encoding a vitamin K-dependent protein. The latter may contain a propeptide sequence other than that which is usually associated with a vitamin K-dependent protein. Expression of a polynucleotide encoding a vitamin K-dependent protein can be obtained either by transfection of a gene of interest into a cell or by activating (i.e., including) an endogenous gene encoding a vitamin K-dependent protein already present in primary, secondary, or immortalized cells taken from vertebrates, which is usually not expressed in cells or not expressed in cells at physiologically significant levels in the form in which these cells are obtained. To activate the genes of interest, homologous recombination can be used to replace or turn off the regulatory region, usually associated with the gene, in the cells as they are obtained, using the regulatory sequence that causes gene expression at levels higher than this is manifested in the corresponding untransfected cell, or for the manifestation of a regulatory or induction structure that is different from that of the corresponding untransfected cell.

In a first aspect, the present invention relates to a eukaryotic host cell expressing a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and expressing a second polynucleotide encoding a second free propeptide, in a second expression unit. You must understand that the first polynucleotide is located in the first expression unit and that the second polynucleotide is located in the second expression unit, where the first and second expression units are different.

In a second aspect, the present invention relates to a eukaryotic host cell transfected with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfected with a second polynucleotide encoding a second free propeptide in a second expression unit. You must understand that the first polynucleotide is located in the first expression unit and that the second polynucleotide is located in the second expression unit, where the first and second expression units are different. The real order of transfection is, of course, trivial, and thus the host cell can be transfected first with a second polynucleotide or vice versa. The use of the terms “first propeptide” and “second propeptide” is for convenience only, thus the first and second propeptides may be the same or different.

The term "eukaryotic host cell", as used here, represents any cell, including hybrid cells into which heterologous DNA can be expressed. Typical host cells include, but are not limited to, insect cells, yeast cells, mammalian cells, including human cells, such as BHK, CHO, HEK and COS cells. In the practice of the present invention, host cells that are cultured are preferably mammalian cells, more preferably an established mammalian cell line, including, but not limited to, CHO cell lines (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), hamster cub kidneys (BHK) and HEK 293 (e.g. ATCC CRL 1573; Graham et al., J. Gen. Virol. 36: 59-72, 1977).

A preferred BHK cell line ^{is a} tH ^- ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982), hereinafter referred to as BHK 570 cells. BHK 570 cell line is available from American type culture collection, 12301 Parklawn Dr., Rockville, MD 20852, accession number ATCC CRL 10314. The BHK tk ^- ts13 cell line is also available from ATCC under accession number CRL 1632.

Other suitable cell lines include, without limitation, Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lungs (ATCC HB 8065 ), NCTC 1469 (ATCC CCL 9.1) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980). Also suitable are 3T3 cells, Namalwa cells, myelomas, and fusion products of myelomas with other cells.

The term "polynucleotide" means a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases, counted from 5 'to the end of 3'. Polynucleotides include RNA and DNA and can be isolated from natural sources, synthesized in vitro, or obtained from a combination of natural and synthetic molecules. The length of the polynucleotide molecule is given here as the number of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term "nucleotides" is used for both single and double-stranded molecules, when the context allows it. When the term is applied to double-stranded molecules, it is used to denote the total length and is understood as the equivalent of the term “base pair”. One of ordinary skill in the art will notice that the two strands of a double stranded polynucleotide may slightly differ in length and that their ends may not coincide with each other as a result of enzymatic cleavage; thus, not all nucleotides within a double-stranded polynucleotide molecule can be paired. Such unpaired ends, as a rule, do not exceed 20 nt in length.

The term "propeptide", as used here, represents any amino acid sequence that can bind gamma-glutamyl carboxylase. Typical propeptides that control the gamma-carboxylation of vitamin K-dependent proteins are found at the N-terminus of the vitamin K-dependent protein and serve as a docking site or recognition sequence for interaction with gamma-glutamylcarboxylase, which carboxylates the glutamate residues typically located in the Gla domain Vitamin K-dependent proteins. More than one binding site for gamma glutamyl carboxylase, i.e., a gamma glutamyl carboxylase recognition sequence, can exist in a single propeptide. One example of a propeptide falling within this definition is thus the sequence of the naturally occurring FVII propeptide. Another example falling within this definition is the sequence of the naturally occurring FVII propeptide linked to the sequence of the natural factor IX propeptide within the same amino acid sequence.

The term “free propeptide,” as used herein, is intended to mean a propeptide that is not bound to a vitamin K-dependent protein that must be gamma-carboxylated. An example of a free propeptide is thus an FVII propeptide that is not linked to the FVII amino acid sequence.

The terms “factor VII” or “FVII,” as used herein, mean a product consisting of an inactive form (factor VII). The terms “factor VIIa” or “FVIIa,” as used herein, mean a product consisting of an activated form (factor VIIa). This includes proteins that have the amino acid sequence 1-406 of native human factor FVII or FVIIa. This also includes proteins with a slightly modified amino acid sequence, for example, a modified end with an N-terminus, including the addition or elimination of amino acids of the N-terminus, insofar as these proteins essentially retain FVIIa activity. "FVII" or "FVIIa", falling under this definition, also includes natural allelic variations that can exist and move from one individual to another. In addition, the degree and location of glycosylation or other post-translational modifications may vary depending on the selected host cells and the nature of the environment for the host cells.

The term "variants", as used here, is intended to mean FVII, where one or more amino acid residues of the source protein are replaced by another amino acid residue, and / or where one or more amino acid residues of the source protein are eliminated, and / or where one or more amino acid residues are added to the original protein. Such an addition can take place either at the N-terminus, or at the C-terminus of the parent protein, or both.

The term “expression unit”, as used herein, means a polynucleotide containing the following elements connected by their action: (a) a transcription promoter; (b) a polynucleotide sequence encoding an amino acid sequence; and (c) a transcription terminator. An example of an expression unit, therefore, is a DNA vector containing the following linked elements: (a) a transcription promoter, (b) a cDNA sequence encoding a free propeptide; and (c) a transcription terminator.

The term “vector,” as used herein, means any nucleic acid object capable of amplification in a host cell. Thus, the vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal object whose replication is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the genome of the host cell and replicates along with the chromosome (s) into which it integrates. The choice of vector often depends on the host cell into which it is to be introduced. Vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, or cosmids. Vectors usually contain a replicator and at least one breeding gene, that is, a gene that encodes a product that is easily detectable or whose presence is essential for cell growth.

The term "promoter" means a part of a gene containing DNA sequences that provide for the binding of RNA polymerase and the initiation of transcription. Promoter sequences are often, but not always, found in 5 'non-coding regions of genes.

In a third aspect, the present invention relates to a eukaryotic host cell expressing a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and expressing a second polynucleotide encoding a second free propeptide in a second expression unit.

In an additional aspect, the present invention relates to a eukaryotic host cell transfected with a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and transfected with a second polynucleotide encoding a second free propeptide in a second expression unit.

The term "vitamin K-dependent protein," as used herein, means any protein that is gamma-carboxylated at glutamic acid residues. Typical vitamin K-dependent proteins include, but are not limited to, procoagulant factors: thrombin, factor VII, IX, and X; anticoagulants: protein C and protein S and other proteins such as osteocalcin (Gla bone protein), matrix Gla protein and proline-enriched protein Gla 1.

In an additional aspect, the present invention relates to a method for producing FVII or its variants, comprising a) expressing in a eukaryotic cell the first polynucleotide encoding the first propeptide and FVII or its variants in the first expression unit and expressing the second polynucleotide encoding the second free propeptide in the second unit expression to produce a coexpressing eukaryotic host cell; b) culturing in an appropriate culture medium a coexpressing eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In an additional aspect, the present invention relates to a method for producing FVII or its variants, comprising a) expressing in a eukaryotic cell the first polynucleotide encoding the first propeptide and FVII or its variants in the first expression unit and expressing the second polynucleotide encoding the second free propeptide in the second unit expression to produce a coexpressing eukaryotic host cell; b) culturing in an appropriate culture medium a coexpressing eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating FVII or variants thereof from the medium.

In an additional aspect, the present invention relates to a method for producing FVII or variants thereof, comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfecting a second polynucleotide encoding a second free propeptide, in a second an expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a co-transfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In an additional aspect, the present invention relates to a method for producing FVII or variants thereof, comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second an expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating FVII or variants thereof from the medium.

In an additional aspect, the present invention relates to a method for producing FVIIa or variants thereof, comprising a) transfection of a eukaryotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfection with a second polynucleotide encoding a second free propeptide, in a second an expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolation and activation of FVII or its variants from the environment.

In a further aspect, the present invention relates to a recombinant vector, wherein said vector comprises a polynucleotide encoding a free propeptide in an expression unit. The embodiments described below with respect to the second polynucleotide, the second free propeptide and the second expression unit are predefined, individually or in combination, also to represent embodiments of the polynucleotide, free propeptide and expression unit contained in a recombinant vector.

In a further aspect, the present invention relates to a recombinant vector, wherein said vector comprises a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and a second polynucleotide encoding a second free propeptide, in a second expression unit.

In a further aspect, the present invention relates to a recombinant vector, wherein said vector comprises a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and a second polynucleotide encoding a second free propeptide in a second expression unit.

In an additional aspect, the present invention relates to a method for producing an eukaryotic host cell producing FVII, comprising a) gene activation in the eukaryotic host cell of the first polynucleotide encoding the amino acid sequence from -18 to 406, FVII and its propeptide, in the first expression unit and b ) transfection with a second polynucleotide encoding a second free propeptide in a second expression unit. In this regard, the amino acid sequence from -18 to -1 is identical to the FVII propeptide identified as SEQ ID NO: 7.

In an additional aspect, the present invention relates to a method for producing an eukaryotic host cell producing FVII or variants thereof, comprising a) transfecting the eukaryotic host cell with a first polynucleotide encoding the first propeptide and FVII or variants thereof, in the first expression unit and b) transfection with the second polynucleotide encoding a second free propeptide in a second expression unit.

In an additional aspect, the present invention relates to a method for producing a vitamin K-dependent protein, comprising a) expressing in a eukaryotic host cell a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and expressing a second polynucleotide encoding a second free propeptide , in a second expression unit to produce a coexpressing eukaryotic host cell; b) culturing in an appropriate culture medium a coexpressing eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In an additional aspect, the present invention relates to a method for producing a vitamin K-dependent protein, comprising a) expressing in a eukaryotic host cell a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and expressing a second polynucleotide encoding a second free propeptide , in a second expression unit to produce a coexpressing eukaryotic host cell; b) culturing in an appropriate culture medium a coexpressing eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating the vitamin K-dependent protein from the medium.

In an additional aspect, the present invention relates to a method for producing a vitamin K-dependent protein, comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a co-transfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In an additional aspect, the present invention relates to a method for producing a vitamin K-dependent protein, comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating the vitamin K-dependent protein from the medium.

In a further aspect, the present invention relates to recombinant FVII or variants thereof obtained by a method comprising a) transfecting a eukarotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a co-transfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In a further aspect, the present invention relates to recombinant FVII or variants thereof obtained by a method comprising a) transfecting a eukarotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating FVII or variants thereof from the medium.

In a further aspect, the present invention relates to recombinant FVIIa or variants thereof obtained by a method comprising a) transfecting a eukarotic host cell with a first polynucleotide encoding a first propeptide and FVII or variants thereof, in a first expression unit and transfecting with a second polynucleotide encoding a second free a propeptide, in a second expression unit, to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolation and activation of FVII or its variants from the environment.

In an additional aspect, the present invention relates to a recombinant vitamin K-dependent protein obtained by a method comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit, to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a co-transfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide.

In an additional aspect, the present invention relates to a recombinant vitamin K-dependent protein obtained by a method comprising a) transfecting a eukaryotic host cell with a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and transfecting with a second polynucleotide encoding a second free propeptide, in a second expression unit, to produce a co-transfected eukaryotic host cell; b) culturing in a suitable culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolating the vitamin K-dependent protein from the medium.

The first propeptide may be such a propeptide, usually associated with a vitamin K-dependent protein, or it may be any other propeptide, such as a propeptide, usually associated with another vitamin K-dependent protein.

In one embodiment, the first propeptide contains one binding sequence for gamma-glutamyl carboxylase. In another embodiment, the first propeptide contains two or more binding sequences for gamma-glutamyl carboxylase. An example of a first propeptide is thus the propeptides commonly associated with FVII and with prothrombin covalently attached to the same expression unit.

In a further embodiment, the second free propeptide comprises one binding sequence for gamma-glutamyl carboxylase. In a further embodiment, the second free propeptide comprises two or more binding sequences for gamma-glutamyl carboxylase. An example of a second free propeptide is thus the propeptides commonly associated with FVII and prothrombin covalently attached to the same expression unit.

In a further embodiment of the present invention, the eukaryotic host cell expresses a first polynucleotide encoding a first propeptide and FVII in a first expression unit and expresses a second polynucleotide encoding a second free propeptide in a second expression unit. In a specific embodiment, FVII is human FVII.

In a further embodiment of the present invention, the eukaryotic host cell is transfected with the first polynucleotide encoding the first propeptide and FVII in the first expression unit and transfected with the second polynucleotide encoding the second free propeptide in the second expression unit. In a specific embodiment, FVII is human FVII.

In a further embodiment of the present invention, the vitamin K-dependent protein is independently selected from prothrombin, factor IX, FVII, factor X, protein C, protein S, osteocalcin rich in proline Gla 1 protein or Gla matrix protein. You must understand that any of these proteins constitutes an alternative embodiment of the present invention.

In a further embodiment of the present invention, the eukaryotic host cell is further transfected with a polynucleotide encoding gamma glutamyl carboxylase in an expression unit. This expression unit may be an expression unit other than the first or second expression unit, or a polynucleotide encoding gamma-glutamyl carboxylase may be located in the first or second expression unit. Examples of gamma glutamyl carboxylases are selected from recombinant human, rat, Drosophila, gamma glutamyl carboxylase, mouse or hamster muscle carboxylase.

In an additional embodiment of the present invention, the first propeptide contains the amino acid sequence of the formula:

X ₁ X ₂ FX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ ,

where X ₁ , X ₄ , X ₅ , X ₆ , X ₇ , X ₉ , X ₁₀ and X ₁₃ are independently selected from

G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T

and where X ₂ , X ₃ , X ₁₁ and X ₁₂ are independently selected from

V, L, P, N, A, I, S, F, M, W, Q, T and Y,

and where X ₈ is independently selected from

G and A

and where X ₁₄ , X ₁₅ , X ₁₆ and X ₁₇ are independently selected from

R, H, A, T, W, L, I, V, Q, K, Y, P or absent.

In one embodiment, a first propeptide X ₁ is selected from A, S, N, R, T and H.

In a further embodiment of the first propeptide X ₂ is selected from V, L, P, N and A.

In a further embodiment of the first propeptide, X ₃ is selected from L, I, V, and S.

In a further embodiment of the first propeptide, X ₄ is

selected from S, R, N, T, D, and A.

In a further embodiment of the first propeptide, X ₅ is selected from R, Q, K, G, H, S, and P.

In a further embodiment of the first propeptide, X ₆ is selected from E, R, and Q.

In a further embodiment of the first propeptide X ₇ is selected from Q, N, E, K and R.

In a further embodiment of the first propeptide, X ₈ is selected from A and G.

In a further embodiment of the first propeptide X ₉ is selected from N, H, S and R.

In a further embodiment of the first propeptide, X ₁₀ is selected from Q, N, T, G, S, K, and E.

In a further embodiment of the first propeptide, X ₁₁ is selected from V, I, F, and L.

In a further embodiment of the first propeptide X ₁₂ is selected from L, I and V.

In a further embodiment of the first propeptide, X ₁₃ is selected from Q, A, S, H, V, K, N, and R.

In a further embodiment of the first propeptide, X ₁₄ is selected or absent from R, H, K, I, and P.

In a further embodiment of the first propeptide, X ₁₅ is selected from R, H, K, Q, V, Y, P, A, T, W, and L or is absent.

In an additional embodiment of the first propeptide, X ₁₆ is selected from R, H, T, Q, K, Y, and P or is absent.

In a further embodiment of the first propeptide, X ₁₇ is selected or absent from R, H and K.

In a further embodiment of the first propeptide, X ₁₇ is R.

In the present description, amino acid residues are presented using abbreviations, as indicated in table 1, approved by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN). With respect to amino acids and the like having isomers, those represented by the following abbreviations are in the natural L-form. Further, the left and right ends of the amino acid sequence of the peptide are N- and C-terminations, respectively, unless otherwise indicated.

In a further embodiment of the present invention, the first propeptide comprises an amino acid sequence with an inhibition constant (Ki) of less than 1 mM. In a further embodiment, the amino acid sequence has a Ki less than 0.5 mm. In yet another embodiment, the amino acid sequence has a Ki ranging between 0.1 nm and 0.5 mm.

In an additional embodiment of the present invention, the first propeptide contains an amino acid sequence of at least 30% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment, the first propeptide comprises an amino acid sequence of at least 40% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment, the first propeptide comprises an amino acid sequence of at least 50% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment of the present invention, the first propeptide comprises an amino acid sequence with a Ki of less than 1 mM and at least 30% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

In an additional embodiment of the present invention, the first propeptide contains a specific amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17 and 18.

In an additional embodiment of the present invention, the second free propeptide contains the amino acid sequence of the formula:

X ₁ X ₂ FX ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ ,

wherein X _1, X _4, X _5, X _6, X _7, X _9, X ₁₀ and X ₁₃

are independently selected from

G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T

and where X ₂ , X ₃ , X ₁₁ and X ₁₂ are independently selected from

V, L, P, N, A, I, S, F, M, W, Q, T and Y,

and where X ₈ is independently selected from

G and A

and where X ₁₄ , X ₁₅ , X ₁₆ and X ₁₇ are independently selected from

R, H, A, T, W, L, I, V, Q, K, Y, P or absent.

In one embodiment of the second free propeptide, X ₁ is selected from A, S, N, R, T, and H.

In a further embodiment of the second free propeptide, X ₂ is selected from V, L, P, N, and A.

In a further embodiment of the second free propeptide, X ₃ is selected from L, I, V, and S.

In a further embodiment of the second free propeptide, X ₄ is selected from S, R, N, T, D, and A.

In a further embodiment of the second free propeptide, X ₅ is selected from R, Q, K, G, H, S, and P.

In a further embodiment of the second free propeptide, X ₆ is selected from E, R, and Q.

In a further embodiment of the second free propeptide, X ₇ is selected from Q, N, E, K, and R.

In a further embodiment of the second free propeptide, X ₈ is selected from A and G.

In a further embodiment, the second free propeptide X ₉ is selected from N, H, S and R.

In a further embodiment of the second free propeptide, X ₁₀ is selected from Q, N, T, G, S, K, and E.

In a further embodiment of the second free propeptide, X ₁₁ is selected from V, I, F, and L.

In a further embodiment of the second free propeptide, X ₁₂ is selected from L, I, and V.

In a further embodiment of the second free propeptide, X ₁₃ is selected from Q, A, S, H, V, K, N, and R.

In a further embodiment of the second free propeptide, X ₁₄ is selected or absent from R, H, K, I, and P.

In a further embodiment of the second free propeptide, X ₁₅ is selected from R, H, K, Q, V, Y, P, A, T, W, and L or is absent.

In a further embodiment of the second free propeptide, X ₁₆ is selected or absent from R, H, T, Q, K, Y, and P.

In a further embodiment of the second free propeptide, X ₁₇ is or is absent from R, H, T, Q, K, Y, and P.

In a further embodiment of the present invention, the second free propeptide comprises an amino acid sequence with a Ki of less than 1 mM. In a further embodiment, the amino acid sequence has a Ki less than 0.5 mM. In yet another embodiment, the amino acid sequence has a Ki ranging between 0.1 nM and 0.5 mM.

In an additional embodiment of the present invention, the second propeptide contains an amino acid sequence of at least 30% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment, the second propeptide comprises an amino acid sequence of at least 40% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment, the second propeptide comprises an amino acid sequence of at least 50% homology with a sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment of the present invention, the second free propeptide comprises an amino acid sequence with a Ki of less than 1 mM and at least 30% homology with a particular sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18.

In an additional embodiment of the present invention, the second free propeptide contains a specific amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

In a further embodiment of the present invention, the eukaryotic host cell is a yeast cell.

In a further embodiment of the present invention, the eukaryotic host cell is an insect cell.

In a further embodiment of the present invention, the eukaryotic host cell is a mammalian cell.

In a further embodiment of the present invention, the eukaryotic host cell is a human cell.

In a further embodiment of the present invention, the eukaryotic host cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells.

In a further embodiment of the present invention, the first polynucleotide is DNA.

In a further embodiment of the present invention, the second polynucleotide is DNA.

In a further embodiment of the present invention, the first polynucleotide is RNA.

In a further embodiment of the present invention, the second polynucleotide is RNA.

In a further embodiment of the present invention, the first expression unit is present on the first vector and the second expression unit is present on the second separate vector.

In a further embodiment of the present invention, the first expression unit and the second expression unit are present on the same vector.

In a further embodiment of the present invention, the first vector is a plasmid vector.

In a further embodiment of the present invention, the second vector is a plasmid vector.

In a further embodiment of the present invention, the first vector is a phage vector.

In a further embodiment of the present invention, the second vector is a phage vector.

In a further embodiment of the present invention, the culture medium is serum free.

In a further embodiment of the present invention, the first polynucleotide is plasmid DNA.

In a further embodiment of the present invention, the second polynucleotide is plasmid DNA.

The term “binding sequence for gamma-glutamyl carboxylase”, as used herein, means the necessary amino acid residues in a sequence (i.e., in a recognition sequence) for binding or docking, or interaction with gamma-glutamyl carboxylase.

In a further embodiment of the present invention, the first propeptide has a higher affinity (as measured by the inhibition constant (Ki)) for carboxylase than the second free propeptide. There are two separate functions of the propeptides of the vitamin K-dependent protein with respect to carboxylase, substrate binding and regulation of activity, different degrees of affinity and concentration of the covalently attached propeptide and free propeptide can affect the overall ability of the producing cell to carboxylate the recombinant vitamin K-dependent protein. Co-expression of the free propeptide and the covalently attached propeptide and their combinations can increase the production of functional recombinant FVII (rFVII). In order not to inhibit the carboxylation of the newly synthesized Glu-containing vitamin K-dependent protein, it is preferable that the second free propeptide has a lower affinity than the propeptide covalently attached to the vitamin K-dependent protein, which should be gamma-carboxylated.

Ki for free propeptides are determined according to Stanley et al. (Biochemistry, 38, 15681-15687 (1999) and J. Biol. Chem. 274, 16940-16944).

In a further embodiment, the free propeptide to be coexpressed with the vitamin K-dependent protein is preferably selected from the lists shown in Tables 2 and 3:

Table 1
Abbreviations for amino acid residues Amino acid Three letter code Single letter code Glycine Gly G Proline Pro P Alanine Ala A Valine Val V Leucine Leu L Isoleucine Ile I Methionine Met M Cysteine Cys C Phenylalanine Phe F Tyrosine Tyr Y Tryptophan Trp W Histidine His H Lysine Lys K Arginine Arg R Glutamine Gln Q Asparagine Asn N Glutamic acid Glu E Aspartic acid Asp D Serine Ser S Threonine Thr T

table 2
Amino acid sequence of propeptides from vitamin K-dependent proteins and consensus propeptide Minus line item eighteen 17 16 fifteen fourteen 13 12 eleven 10 9 8 7 6 5 four 3 2 one * Ki nM Consensus * A V F L S R E Q A N Q V L Q R R R R 0.43 factor X S L F I R R E Q A N N I L A R V T R 2.6 Matrix Gla Protein N P F I N R R N A N T F I S P Q Q R 5.8 Fvii A V F V T Q E E A H G V L H R R R R 11.1 Protein S A N F L S K Q Q A S Q V L V R K R R 12,2 PRGP1 R V F L T G E K A N S I L K R Y P R 12.8 factor IX T V F L D H E N A N K I L N R P K R 33.6 protein C S V F S S S E R A H Q V L R I R K R 230 Prothrombin H V F L A P Q Q A R S L L Q R V R R 277 Osteocalcin A A F V S K Q E G S E V V K R P R R > 500,000

* Stanley et al. (1999) Biochemistry, 38, 15681-15687 and J. Biol. Chem. 274, 16940-16944

Table 3
Examples of free propeptides that should be coexpressed with a vitamin K-dependent protein Minus line item eighteen 17 16 fifteen fourteen 13 12 eleven 10 9 8 7 6 5 four 3 2 one Authentic Fvii A V F V T Q E E A H G V L H R R R R Factor IX T V F L D H E N A N K I L N R P K R Protein C S V F S S S E R A H Q V L R I R K R Mutants Osteocalcin (G-10A, V-6L) A A F V S K Q E A S E V L K R P R R FVII (A-10G) A V F V T Q E E G H G V L H R R R R FVII (H-9R) A V F V T Q E E A R G V L H R R R R FVII (H-9K) A V F V T Q E E A K G V L H R R R R FVII (H-9L) A V F V T Q E E A L G V L H R R R R FVII (V-15S) A V F S T Q E E A H G V L H R R R R Processed Fvii A V F V T Q E E A H G V L H

Factor IX T V F L D H E N A N K I L N Protein C S V F S S S E R A H Q V L R

The present invention also relates to a method for producing vitamin K-dependent proteins, as discussed above. Vitamin K-dependent proteins are preferably obtained using recombinant DNA techniques. From this point of view, DNA sequences encoding vitamin K-dependent proteins can be isolated by preparing a genomic or cDNA library and looking at DNA sequences encoding all or part of the protein by hybridization using synthetic oligonucleotide samples in accordance with standard techniques (see, Sambrook et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989). For this purpose, the DNA sequence encoding the protein should preferably be derived from a human, that is, obtained from a genomic DNA library or human cDNA library.

The present invention also relates to a method for activating (i.e., incorporating) a gene encoding a vitamin K-dependent protein present in primary, secondary or immortalized cells derived from vertebrates, which is usually not expressed in cells or not expressed in cells at physiologically significant levels in as received. Homologous recombination can be used to replace or deactivate the regulatory region, usually associated with the gene, in the cells, as obtained, with the regulatory sequence, which should cause gene expression at levels higher than what appears in the corresponding untransfected cell, or for the manifestation of a regulatory structure or induction that is different from that which appears in the corresponding untransfected cell. The present invention, therefore, also relates to a method for producing vitamin K-dependent proteins by incorporating or activating an endogenous gene that encodes a vitamin K-dependent protein in transfected primary, secondary or immortalized cells. Activation of endogenous genes can be accomplished as described in US Pat. No. 5,968,502. DNA sequences encoding vitamin K-dependent proteins can also be synthesized using established standard methods, for example, the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981) , 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. In accordance with the phosphoamidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, annealed, ligated and cloned into the corresponding vectors.

DNA sequences can also be obtained by polymerase chain reaction using specific primers, as, for example, described in US patent 4683202, Saiki et al., Science 239 (1988), 487-491, or Sambrook et al., Above.

DNA sequences encoding vitamin K-dependent proteins are typically inserted into a recombinant vector, which can be any vector that is conveniently subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector can be an autonomously replicating vector, that is, a vector that exists as an extrachromosomal object whose replication is independent of chromosomal replication, for example, a plasmid. Alternatively, the vector may be one that, when introduced into the host cell, integrates into the genome of the host cell and replicates along with the chromosome (s) into which it integrates.

The vector is preferably an expression vector in which the DNA sequence encoding the vitamin K-dependent proteins, when acted upon, is linked to additional segments required for DNA transcription. Typically, an expression vector is derived from plasmid or viral DNA, or it may contain elements of both of them. The term "linked in action" indicates that the segments are arranged in such a way that they function in accordance with their intended tasks, for example, transcription is initiated in the promoter and is carried out along the DNA sequence encoding the polypeptide.

The promoter may be any DNA sequence that exhibits transcriptional activity in a selected host cell and can be obtained from genes encoding proteins, either homologous or heterologous to the host cell.

Examples of suitable promoters for controlling the transcription of DNA encoding a vitamin K-dependent protein in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. 1 (1981), 854-864), the MT-1 promoter (metallothionein gene ) (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41: 521-530, 1985) or the main late promoter of adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol, 2: 1304-1319, 1982).

An example of a suitable promoter for use in insect cells is the polyhedrin promoter (US Pat. No. 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (JM Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the Autographa californica polyhedrosis virus main protein promoter (European patent EP 397485), the immediate early promoter of the baculovirus 1 gene (US patent 5155037; US patent 5162222) or the delayed early promoter of the 39K baculovirus gene (US patent 5,155,037; U.S. Patent 5,162,222).

Examples of suitable promoters for use in yeast host cells include promoters from glycolytic yeast genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen . 1 (1982), 419-434) or the gene for alcohol dehydrogenase (Young et al., In Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or TPI1 promoters (US patent 4599311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654).

Examples of suitable promoters for use in filamentous fungal host cells are, for example, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Examples of other useful promoters are those derived from a gene encoding A.oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A.niger neutral α-amylase, A.niger stable acid α-amylase, glucoamylase (gluA) A .niger or A.awamori, Rhizomucor miehei lipase, A.oryzae alkaline protease, A.oryzae triosophosphate isomerase or A.nidulans acetamidase. TAKA amylase and gluA promoters are preferred. Appropriate promoters are discussed, for example, in European patent EP 238023 and European patent EP 383779.

DNA sequences encoding vitamin K-dependent proteins can also, if necessary, be combined with an appropriate terminator, such as a human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814), or terminators, if necessary. TPI1 (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or ADH3 (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099). The vector may also contain a set of spliced RNA regions located after the promoter and before the insertion site of sequence VII itself. Preferred spliced RNA regions may be derived from adenovirus and / or immunoglobulin genes. The expressed vectors also contain a polyadenylation signal located after the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), The polyadenylation signal from the Elb region of adenovirus 5, the terminator of the human growth hormone gene (DeNoto et al., Nuc. Acids Res. 9: 3719 -3730, 1981) or a polyadenylation signal from a human FVII gene or a bovine FVII gene. Expressed vectors may also include a non-coding leader sequence of the virus, such as a triple leader sequence of adenovirus 2 located between the spliced regions of the promoter and RNA; and enhancer sequences, such as the SV40 enhancer sequence.

The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell of interest. An example of such a sequence (when the host cell is a mammalian cell) is an SV40 replicator.

When the host cell is a yeast cell, the corresponding sequences enabling the vector to replicate are replication genes of the 2μ REP 1-3 yeast plasmid and replicators.

The vector may also contain a selectable marker, for example a gene whose product is complementary to a defect in the host cell, such as a gene encoding a dihydrofoliate reductase (DHFR) or TPI gene Schizosaccharomyces pombe (described by PR Russell, Gene 40, 1985, pp. 125-130 ), or a gene that is responsible for drug resistance, such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. For filamentous fungi, selectable markers include amdS, pyrG, argB, niaD or sC.

To direct the vitamin K-dependent proteins of the present invention into the secretory pathway of host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre-sequence) can be provided in a recombinant vector. The secretory signal sequence combines with DNA sequences encoding vitamin K-dependent proteins in the correct reading frame. Secretory signal sequences are often located at position 5 'with respect to the DNA sequence encoding the peptide. The secretory signal sequence may be a sequence usually associated with a protein, or may be taken from a gene encoding another secreted protein.

For secretion from yeast cells, the secretory signal sequence can encode any signal peptide that provides for the efficient directing of expressed vitamin K-dependent proteins to the secretory pathway of the cell. The signal peptide may be a natural signal peptide or a functional part thereof, or it may be a synthetic peptide. Suitable signal peptides for use are found to be the α-factor signal peptide (see US Pat. No. 4,871,000), the mouse saliva amylase signal peptide (see, O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646 ), a modified carboxypeptidase signal peptide (see LA Vails et al., Cell 48, 1987, pp. 887-897), BAR1 yeast signal peptide (see European patent application WO 87/02670) or yeast asparagin protease signal peptide 3 (YAP3) (see, M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

For effective secretion in yeast, the sequence encoding the leader peptide can also be inserted after the signal sequence and before the DNA sequence encoding the vitamin K-dependent proteins. The function of the leader peptide is to allow the expressed peptide to be directed from the endoplasmic reticulum to the Golgi apparatus and then to the secretory vesicle for secretion into the culture medium (i.e., to isolate vitamin K-dependent proteins through the cell wall or, at least, through the cell wall membrane into the periplasmic space of a yeast cell). The leader peptide may be the yeast α-factor leader sequence (the use of which is described, for example, in US Pat. No. 4,546,082, US Pat. No. 4,871,000, EP 16201, EP 123294, EP 123544 and EP 163529). Alternatively, the leader peptide may be a synthetic leader peptide, which, so to speak, is a leader peptide that is not found in nature. Synthetic leader peptides can, for example, be constructed in the manner described in European patent application WO 89/02463 or in European patent application WO 92/11378.

For use in filamentous fungi, a signal peptide is conveniently derived from a gene encoding Aspergillus sp. Amylase or glucoamylase, a gene encoding a Rhizomucor miehei lipase or protease, or Humicola lanuginosa lipase. Preferably, the signal peptide is derived from a gene encoding A.oryzae TAKA amylase, A.niger neutral α-amylase, A.niger stable acid amylase, or A.niger glucoamylase. Corresponding signal peptides are described, for example, in European patent EP 238023 and in European patent EP 215594.

For use in insect cells, a signal peptide is conveniently derived from an insect gene (see European patent application WO 90/05783), such as a signal peptide of the precursor of the adipokinetic hormone of Lepidoptera Manduca sexta (see US Pat. No. 5,023,328).

The procedures used to ligate DNA sequences encoding vitamin K-dependent proteins, a promoter and, optionally, a terminator and / or secretory signal sequence, respectively, and how to insert them into appropriate vectors containing information necessary for replication, are well known to specialists in this field (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

Methods for transfecting mammalian cells and expressing DNA sequences introduced into cells are described, for example, in Kaufman and Sharp, J. Mol. Biol. 159 (1982), 601-621; Southern and Berg. J. Mol. Appl. Genet. 1 (1982). 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603, Graham and van der Eb. Virology 52 (1973), 456; and Neumann et al., EMBO J. 1 (1982), 841-845.

Selectable markers can be introduced into the cell on a separate plasmid simultaneously with the gene of interest, or they can be introduced on the same plasmid. If they are introduced on the same plasmid, the selectable marker and the gene of interest can be controlled by different promoters or the same promoter, the latter structure produces a two-cistron signal. Designs of this type are known in the art (e.g., Levinson and Simonsen, US Patent No. 4,713,339). It may also be advantageous to add additional DNA, known as a “carrier DNA," to the mixture that is introduced into the cells.

After the cells absorb the DNA, they are grown in an appropriate growth medium, usually 1-2 days, so that they begin to express the gene of interest. As used herein, the term “appropriate growth medium” means a medium containing nutrients and other components required for cell growth and expression of a vitamin K-dependent protein of interest. Media typically include a carbon source, a nitrogen source, basic amino acids, basic sugars, vitamins, salts, phospholipids, protein and growth factors. To produce gamma-carboxylated proteins, the medium must contain vitamin K, preferably at a concentration of from about 0.1 μg / ml to about 5 μg / ml. Drug selection is then applied to select the growth of cells that express the selectable marker in a stable manner. For cells that are transfected with an amplifying selectable marker, the drug concentration can be increased to select an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then scanned for expression of the vitamin K-dependent protein of interest.

A host cell into which DNA sequences encoding vitamin K-dependent proteins are introduced can be any cell that is capable of producing post-translationally modified vitamin K-dependent proteins, and includes yeast, fungal, and higher eukaryotic cells.

Examples of mammalian cell lines for use in the present invention are COS-1 (ATCC CRL 1650), Hamster Baby Kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. Cell lines: 36: 59- 72, 1977). A preferred BHK cell line is the BHK cell line tk.sup.-ts13 (Waechterand Baserga, Proc. Natl. Acad. Sci. USA 79: 1106-1110, 1982, incorporated herein by reference), hereinafter referred to as BHK 570 cells. The BHK 570 cell line is in the American Type Culture Collection, 12301 Parklawn Dr., Rockville, Md. 20852, under ATCC accession number CRL 10314. The BHK cell line tk.sup.-ts13 is also available under ATCC accession number CRL 1632. In addition, a number of cell lines can be used in the present invention, including Rat Hep I (rat hepatoma; ATCC CRL 1600), Rat Hep II (rat hepatoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lungs (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), CHO (ATCC CCL 61) and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216-4220, 1980).

Examples of usable yeast cells include Saccharomyces spp cells. or Schizosaccharomyces spp. in specific strains of Saccharomyces cerevisiae or Saccharomyces kluyveri. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides are described, for example, in US Pat. No. 4,599,311, US Pat. No. 4,931,373, US Pat. No. 4,871,000, 5,073,743 and US Pat. No. 4,845,075, all of which are incorporated herein by reference. Transformed cells are selected according to the phenotype determined by the selectable marker, often by drug resistance or ability to grow in the absence of a specific nutrient, such as leucine. A preferred vector for use in yeast is the POT1 vector described in US Pat. No. 4,931,373. DNA sequences encoding vitamin K-dependent proteins may be preceded by a signal sequence and, optionally, a leader sequence, as described above. Other examples of suitable yeast cells are Kluyveromyces strains such as K. lactis, Hansenula, for example H. polymorpha, or Pichia, for example P. pastoris (see Gleeson et al., J. Gen. Microbiol. 132, 1986, p. 3459-3465; U.S. Patent 4,882,279).

Examples of other fungal cells are filamentous fungal cells, for example Aspergillus spp., Neurospora spp., Fusarium spp. or Trichoderma spp., in particular strains of A.oryzae, A.nidulans or A.niger. Using Aspergillus spp. for the expression of proteins is described, for example, in European patent EP 272277, European patent EP 238023, European patent EP 184438. The transformation of F. oxysporum can be carried out, for example, as described in Malardier et al., 1989, Gene 78: 147-156. Transformation Trichoderma spp. can be carried out, for example, as described in European patent EP 244234.

When a filamentous fungal cell is used as the host cell, it can be transformed using the DNA construct of the present invention, while it is convenient to integrate the DNA construct into the host chromosome to obtain a recombinant host cell. This integration, in general, is seen as an advantage, since the DNA sequence has a high probability of stable maintenance in the cell. The integration of DNA constructs into the host chromosome can be carried out in accordance with conventional methods, for example, by homologous or heterologous recombination.

Transformation of insect cells and the production of heterologous polypeptides in them can be carried out as described in US patent 4745051; U.S. Patent 4,879,236; U.S. Patent 5,155,037; 5,162,222; in European patent EP 397485; all are included here as links. The insect cell line used as a host may suitably be a Lepidoptera cell line, such as Spodoptera frugiperda cells or Trichoplusia ni cells (see US Pat. No. 5,077,214). Cultivation conditions may suitably be as described, for example, in European patent applications WO 89/01029 or WO 89/01028 or in any of the above references.

The transformed or transfected host cell described above is then cultured in an appropriate nutrient medium under conditions enabling expression of the vitamin K-dependent protein, after which part of the obtained peptide (or all) of it can be recovered from the culture. The medium used for culturing cells may be any conventional medium suitable for use in growing host cells, such as minimal or complex media containing appropriate excipients. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g., catalogs of the American Type Culture Collection). The vitamin K-dependent protein produced by the cells can then be recovered from the culture medium using conventional procedures, including isolating the host cells from the medium by centrifugation or filtration, precipitating the protein components of the supernatant or filtrate by means of a salt, for example ammonium sulfate, by purification using various chromatographic procedures, for example, ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, depending on the type of polypeptide in question .

To obtain recombinant human FVII or variants thereof, a cloned wild-type FVII DNA sequence is used. This sequence may be modified to encode the desired FVII protein or variants thereof. This sequence is then inserted into an expressed vector, which, in turn, is transformed or transfected into host cells. Higher eukaryotic cells, in particular cultured mammalian cells, are preferred as host cells. Complete nucleotide and amino acid sequences for human FVII are known. See US Patent No. 4,784,950, which is incorporated herein by reference for cloning and expression of recombinant human FVII. The bovine FVII sequence is described in Takeya et al., J. Biol. Chem, 263: 14868-14872 (1988), which is incorporated herein by reference.

Changes in amino acid sequence can be obtained using various methods. DNA sequence modification may be site-specific mutagenesis. Site-specific mutagenesis techniques are well known in the art and are described, for example, by Zoller and Smith (DNA 3: 479-488, 1984). Thus, using the nucleotide and amino acid sequences of FVII, you can enter the desired changes.

DNA sequences for use in the present invention will typically encode the prepropeptide at the amino terminus of the FVII protein to obtain appropriate post-translational processing (e.g., gamma-carboxylation of glutamic acid residues) and secretion from the host cell. The prepropeptide may be a peptide from FVII or another vitamin K-dependent plasma protein, such as factor IX, factor X, prothrombin, protein C or protein S. As will be appreciated by one of skill in the art, further modifications to the FVII amino acid sequence can be made there where such modifications do not significantly impair the ability of the protein to act as a coagulation factor. For example, FVII in a catalytic triad can also be modified at a cleavage activation site to inhibit the conversion of zymogen FVII to its activated double-stranded form, as described in general terms in US Pat. No. 5,288,629, which is incorporated herein by reference.

In the present invention, transgenic animal technology can be used to produce the vitamin K-dependent protein. It is preferred to produce proteins using the mammary glands of a female mammalian host. Expression in the mammary glands and subsequent secretion of the protein of interest in milk overcomes many of the difficulties encountered in isolating proteins from other sources. Milk is easy to collect, available in large quantities and is well characterized biochemically. In addition, the main proteins of milk are present in milk at high concentrations (usually from about 1 to 15 g / l). From a commercial point of view, it is clearly preferable to use species that provide a large yield of milk as a host. Although smaller animals, such as mice and rats, can be used (and are preferred at the stage of basic verification), in the present invention it is preferable to use mammals that make up livestock, including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to factors such as a previous history of transgenesis of this species, milk yield, cost and easy availability of sheep milk collection equipment. See WIPO publication European patent application WO 88/00239 for a comparison of factors influencing the choice of host species. It is generally desirable to select a breed of animal host that has been bred for use as a dairy, such as East Friesland sheep, or for producing dairy cattle by breeding a transgenic line recently. In any case, animals with a known good state of health should be used.

To obtain expression in the mammary gland, a transcription promoter from the milk protein gene is used. Milk protein genes include those genes that encode caseins (see US Pat. No. 5,304,489, incorporated herein by reference), beta-lactoglobulin, α-lactalbumin and whey acid protein. A beta-lactoglobulin promoter (BLG) is preferred. In the case of the sheep beta-lactoglobulin gene, as a rule, a region of at least the nearest 406 bp from the gene sequence flanking 5 ′ should be used, although large portions of the sequence flanking 5 ′ are preferred, up to about 5 kbp, for example, an approximately 4.25 kbp DNA segment spanning the 5 'flanking promoter and a non-coding portion of the beta-lactoglobulin gene. See Whitelaw et al., Biochem J. 286: 31-39 (1992). Similar promoter DNA fragments from other species are also suitable for use.

Other regions of the beta-lactoglobulin gene may also be included in the construct if they may contain genomic regions of the gene to be expressed. In this area, it is generally accepted that constructs where, for example, no introns are present, are poorly expressed compared to those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840 (1988); Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991); European applications patent WO 89/01343 and WO 91/02318, each of which is incorporated herein by reference). In this regard, it is generally preferable, where possible, to use genomic sequences containing all of the native introns or some of them from a gene encoding a protein or polypeptide of interest, thus additional inclusion of at least some introns , for example, from the beta-lactoglobulin gene is preferred. One such region is a DNA segment that allows intron splicing and RNA polyadenylation from the 3 ′ non-coding region of the sheep beta-lactoglobulin gene. When it replaces the natural 3 'non-coding sequences of a gene, this segment of sheep beta-lactoglobulin can both increase and stabilize expression levels of the protein or polypeptide of interest. In other embodiments, the region surrounding the ATG sequence for initiating a sequence encoding a vitamin K-dependent protein is replaced by the corresponding sequences from the gene for a particular milk protein. Such substitution provides a favorable tissue-specific initiation environment to enhance expression. It is convenient to replace the entire prepro sequence of the vitamin K-dependent protein and 5 'non-coding sequences with sequences, for example, of the BLG gene, although smaller regions can be replaced.

For expression of a vitamin K-dependent protein in transgenic animals, a DNA segment encoding a vitamin K-dependent protein is coupled with additional DNA segments required for its expression to create expression units. Such additional segments include the above promoter, as well as sequences that terminate the transcription and polyadenylation of mRNA. The expression units must additionally include a DNA segment encoding a secretory signal sequence associated with a segment encoding a vitamin K-dependent protein. The secretory signal sequence may be a native secretory signal sequence of a vitamin K-dependent protein, or may be the sequence of another protein, such as milk protein. See, for example, von Heinje, Nuc. Acids Res. 14: 4683-4690 (1986) and Meade et al., US Pat. No. 4,873,316, which are incorporated herein by reference.

The construction of expression units for use in transgenic animals is conveniently carried out by inserting a sequence encoding a vitamin K-dependent protein into a plasmid or phage vector containing additional DNA segments, although the expression unit can be constructed using any essentially ligation sequence. It is especially convenient to create a vector containing a DNA segment encoding a milk protein, and replace the coding sequence of a milk protein with a vitamin K-dependent protein sequence, thereby creating a fusion gene that includes control sequences for expressing the milk protein gene. In any case, cloning the expression units into plasmids or other vectors facilitates the amplification of the vitamin K-dependent protein. Amplification is conveniently carried out in bacterial (for example, E. coli) host cells in such a way that the vectors, as a rule, should include the origin of replication and a selectable marker that functions in bacterial host cells.

Then, the expression unit is introduced into the fertilized eggs (including embryos in the early stages of development) of the selected host species. The introduction of heterologous DNA can be carried out using one of several methods, including microinjection (for example, US patent No. 4873191), retroviral infection (Jaenisch, Science 240: 1468-1474 (1988)), or site-oriented integration using embryonic stem (ES) cells ( review by Bradley et al., Bio / Technology 10: 534-539 (1992)). Then the eggs are implanted in the ovaries or uterus of the pseudopregnant females and allowed to develop until a certain time. Offspring carrying DNA in their infected lineage can transfer DNA to their offspring in the usual Mendelian way, making it possible to breed transgenic flocks.

General procedures for breeding transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Biotechnology 6: 179-183 (1988); Wall et al., Biol. Reprod. 32: 645-651 (1985); Buhler et al., Biotechnology 8: 140-143 (1990); Ebert et al., Biotechnology 9: 835-838 (1991); Krimpenfort et al., Bio / Technology 9: 844-847 (1991); Walletal., J. Cell. Biochem. 49: 113-120 (1992); U.S. Patent Nos. 4,873,191 and 4,873,316; WIPO publication of applications for European patents WO 88/00239, WO 90/05188, WO 92/11757 and UK patent GB 87/00458, which are incorporated herein by reference. Techniques for introducing extraneous DNA sequences into mammals and into their infected cells were originally developed for mice. See, for example, Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, Cell 41: 343-345 (1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985) and Hogan et al. (ibid.). Subsequently, these techniques have been adapted for use on larger animals, including various livestock species (see, for example, WIPO publication of European patent applications WO 88/00239, WO 90/05188 and WO 92/11757 and Simons et al., Bio / Technology 6: 179-183 (1988)). As a result, in the most effective method used to date in the generation of transgenic mice or livestock, several hundred linear DNA molecules of interest are injected into one of the nuclei of a fertilized egg in accordance with established procedures. DNA injection into the zygote cytoplasm may also be used. Can also be used production in transgenic plants. Expression can be generalized or directed to a specific organ, such as a tuber. See Hiatt, Nature 344: 469-479 (1990); Edelbaum et al., J. Interferon Res. 12: 449-453 (1992); Sijmons et al., Bio / Technology 8: 217-221 (1990) and EP 255378.

The FVII obtained by the present invention can be purified by affinity chromatography on an anti-FVII antibody column. It is preferred that the immunoadsorption column contains a highly specific monoclonal antibody. Use of calcium dependent monoclonal antibodies as described by Wakabayashi et al., J. Biol. Chem. 261: 11097-11108, (1986) and Thim et al., Biochem. 27: 7785-7793 (1988), which are incorporated herein by reference, is particularly preferred. Additional purification can be achieved using conventional chemical cleaning agents, such as high performance liquid chromatography. Other purification methods are known in the art, including precipitation with barium citrate, and they can be used to purify the FVII described herein (see, in general, Scopes, R., Protein, Purification, Springer-Verlag, N.Y., 1982). For pharmaceutical applications, substantially pure FVII with a homogeneity of at least about 90-95%, and most preferably with a homogeneity of from 98 to 99% or more, is preferred. After purification, partial or homogeneous, if desired, FVII can then be used for therapy.

Conversion of single-stranded FVII to active double-stranded FVIIa can be achieved using factor XIIa as described by Hedner and Kisiel (1983, J. Clin. Invest. 71: 1836-1841), or other proteases having trypsin-like specificity (Kisiel and Fujikawa, Behring Inst. Mitt. 73: 29-42, 1983). Alternatively, FVII can be activated by passing it through an ion exchange chromatographic column, such as mono Q.RTM (Pharmacia Fire Chemicals), or the like (Bjoern et al., 1986, Research Disclosures 269: 564-565). The FVII molecules of the present invention and their pharmaceutical compositions are particularly useful for administration to humans for the treatment of various conditions, including intravascular coagulation.

Vitamin K-dependent proteins of the present invention can be used to treat certain types of hemophilia. Hemophilia A is characterized by the absence of active factor VIII, factor VIIIa or the presence of inhibition of factor VIII. Hemophilia B is characterized by the absence of active factor IX, factor IXa. FVII deficiency, although rare, responds well to the administration of factor VII (Bauer, K. A., 1996, Haemostasis, 26: 155-158, suppl. 1). Factor VIII replacement therapy is limited due to the development of a high titer of inhibitor antibodies to factor VIII in some patients. Alternatively, FVIIa can be used in the treatment of hemophilia A and B. Factor IXa and factor VIIIa activate factor X. Factor VIIa eliminates the need for factors IX and VIII by directly activating factor X and can overcome the deficiency of factor IX and VIII with few immunological consequences.

Brief Description of the Drawings

The present invention is described in more detail in the examples with reference to the accompanying drawings, where

Figure 1. The structure of correctly processed human FVII coagulation, amino acids from 1 to 406, with gamma-carboxylated Glu (γ) residues and glycosylation (*). The arrow on the residue of amino acid 152 shows the site where the single-stranded FVII is cleaved to convert to activated double-stranded FVII (FVIIa).

Figure 2. Construction of plasmids for expression of free propeptides and for expression of recombinant human FVII with attached propeptide. Plasmids pLN171 and pLN174 express human FVII together with the attached propeptide associated with FVII in nature. Plasmid pLN329 contains a stop codon inserted into the cDNA encoding FVII, after the propeptide associated with FVII, in nature, to express only the free propeptide.

Figure 3. Comparison of CHO-K1 cells expressing FVII (control) and cells coexpressing FVII and free FVII propeptide (FVII propeptide). The results are presented in pg of FVII expression per cell per day.

The present invention is further illustrated by the following examples, which, however, should not be construed as limiting the scope protected by the invention. The features described in the above description and in the following examples may, both individually and in any combination thereof, constitute material for implementing the present invention in its various forms.

EXAMPLES

Example 1

Coexpression of FVII and FVII Propeptide in CHO-K1 Cells

The plasmid vector pLN174 for the expression of human FVII has been described (Persson and Nielsen, 1996, FEBS Lett. 385: 241-243). Briefly, it carries the cDNA nucleotide sequence encoding human FVII, including the propeptide, under the control of the mouse metallothionein promoter for transcription of the mouse cDNA and cDNA insert reductase dihydrofoliate mouse under the control of the SV40 early promoter for use as a selectable marker.

To construct a plasmid vector encoding a gamma recognition carboxylation sequence, the pBluescript II KS + cloning vector (Stratagene) containing cDNA encoding FVII, including its propeptide (pLN171) (Persson et al. 1997, J. Biol. Chem. 272: 19919, is used -19924). The nucleotide sequence encoding stopcodon is inserted into the cDNA encoding FVII after the FVII propeptide using reverse PCR-mediated mutagenesis on this cloning vector. The matrix plasmid was denatured by treatment with NaOH followed by PCR with Pwo polymerases (Boehringer-Mannheim) and Taq (Perkin-Elmer) with the following primers:

a) 5'-AGC GTT TTA GCG CCG GCG CCG GTG CAG GAC-3 '(SEQ ID NO: 19)

b) 5'-CGC CGG CGC TAA AAC GCT TTC CTG GAG GAG CTG CGG CC-3 '(SEQ ID NO: 20).

The resulting mixture was digested with DpnI to digest the remainder of the template DNA, and Escherichia coli was transformed using the PCR product. Clones are scanned by sequencing for the presence of a mutation. cDNA from the correct clone is transferred as a BamHI-EcoRI fragment to the pcDNA3 expression plasmid (Invitrogen). The resulting plasmid is designated as pLN329.

CHO K1 cells (ATCC CCI61) are transfected with equal amounts of pLN174 and pLN329 using the Fugene6 method (Boehriner-Mannheim). Transfectants are selected by adding methotrexate up to 1 μM and G-418 up to 0.45 mg / ml. The transfectant pool is cloned by boundary dilution and the expression of FVII from the clones is measured.

The highly productive clone is further subcloned and clone E11 with specific expression of FVII 2.4 pg / cell / day in Dulbecco's modified Eagle medium with 10% fetal calf serum is selected. The clone adapts to a serum-free suspension culture in a commercially available CHO medium (JRH Bioscience).

Adapted cells multiply continuously in agitated cultures and, when the number of cells increases, the volume is gradually increased by adding a new medium.

After 25 days, 6 L of the culture with stirring was inoculated into a 50 liter biological reactor. Cells multiply in a biological reactor, and when the number of cells increases, the volume gradually increases by adding a new medium.

Finally, 50 L of inoculum was inoculated into a 500 liter industrial biological reactor containing macroporous Cytopore 1 carriers (Pharmacia), after which the suspended cells were immobilized on the carriers. The culture is maintained at 36 ° C, at a pH of 7.0-7.1 and at a dissolved oxygen pressure (DOT) of 50% of saturation. The volume in an industrial biological reactor gradually increases by adding a new medium as the number of cells increases. When the cell density reaches approximately 2 × 10 ⁵ cells / ml, the production phase is initiated and the medium is changed every 24 hours: stirring is stopped to allow sedimentation of carriers containing cells, and then 80% of the culture supernatant is collected and replaced with a new medium. The collected culture supernatant is filtered to remove unreached cells (i.e. cells that cannot be immobilized on carriers) and cell debris, and then transported for further processing.

During the production phase, the cell density reaches 2 3 × 10 ⁷ cells / ml and a titer of 8 mg factor VII / liter.

Example 2

Comparison of CHO-K1 cells expressing FVII and cells coexpressing FVII and free FVII propeptide

CHO-K1 cells are transfected with pLN174 and 1) the empty vector pcDNA3.1 + or 2) the FVII propeptide in pcDNA3.1 +, as described in Example 1. The transfectant pools are analyzed for FVII expression and cell number. FVII yields over 24 hours are shown in FIG. The results show that coexpression of the FVII propeptide increases FVII cell yields from about 1 pg / cell / day to 4 pg / cell / day. The results are presented in figure 3.

List of sequences:

SEQ ID NO: 1: AVFLSREQANQVLQRRRR

SEQ ID NO: 2: SLFIRREQANNILARVTR

SEQ ID NO: 3: NPFINRRNANTFISPQQR

SEQ ID NO: 4: ANFLSKQQASQVLVRKRR

SEQ ID NO: 5: RVFLTGEKANSILKRYPR

SEQ ID NO: 6: HVFLAPQQARSLLQRVRR

SEQ ID NO: 7: AVFVTQEEAHGVLHRRRR

SEQ ID NO: 8: TVFLDHENANKILNRPKR

SEQ ID NO: 9: SVFSSSERAHQVLRIRKR

SEQ ID NO: 10: AAFVSKQEASEVLKRPRR

SEQ ID NO: 11: AVFVTQEEGHGVLHRRRR

SEQ ID NO: 12: AVFVTQEEARGVLHRRRR

SEQ ID NO: 13: AVFVTQEEAKGVLHRRRR

SEQ ID NO: 14: AVFVTQEEALGVLHRRRR

SEQ ID NO: 15: AVFSTQEEAHGVLHRRRR

SEQ ID NO: 16: AVFVTQEEAHGVLH

SEQ ID NO: 17: TVFLDHENANKILN

SEQ ID NO: 18: SVFSSSERAHQVLR

SEQ ID NO: 19: 5'-AGC GTT TTA GCG CCG GCG CCG GTG CAG GAC-3 '

SEQ ID NO: 20: 5'-CGC CGG CGC TAA AAC GCT TTC CTG GAG GAG CTG CGG CC-3 '

Claims (38)

1. A eukaryotic host cell expressing the first polynucleotide encoding the first propeptide and FVII or its functional variants in the first expression unit, and expressing the second polynucleotide encoding the second free propeptide in the second expression unit, where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. 2. Expressing eukaryotic host cell transfected with the first polynucleotide encoding the first propeptide and FVII or its functional variants in the first expression unit, and transfected with the second polynucleotide encoding the second free propeptide in the second expression unit, where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 . 3. The eukaryotic host cell according to any one of claims 1 and 2, wherein said first polynucleotide encodes a first human propeptide and FVII. 4. A eukaryotic host cell expressing a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit and expressing a second polynucleotide encoding a second free propeptide in a second expression unit, where each of said first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. 5. Expressing eukaryotic host cell transfected with the first polynucleotide encoding the first propeptide and vitamin K-dependent protein in the first expression unit, and transfected with the second polynucleotide encoding the second free propeptide in the second expression unit, where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 . 6. A eukaryotic host cell according to any one of claims 4 and 5, wherein said first polynucleotide encodes a vitamin K-dependent protein independently selected from prothrombin, factor IX, FVII, factor X, protein C, protein S, osteocalcin rich in protein proline Gla 1 or matrix protein Gla. 7. The eukaryotic host cell according to any one of claims 1 to 6, wherein said host cell is transfected with an additional polynucleotide encoding gamma glutamyl carboxylase in an expression unit. 8. The eukaryotic host cell according to any one of claims 1 to 7, where the specified host cell is a mammalian cell. 9. The eukaryotic host cell of claim 8, wherein said mammalian cell is a human cell. 10. The eukaryotic host cell of claim 9, wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells. 11. A eukaryotic host cell according to any one of claims 1 to 10, wherein said first and second polynucleotides are DNA. 12. The eukaryotic host cell according to any one of claims 1 to 11, wherein said first and second polynucleotides are RNA. 13. A method of obtaining FVII or its functional variants, including a) transfection of the eukaryotic host cell with the first polynucleotide encoding the first propeptide and FVII or its functional variants in the first expression unit, and transfection with the second polynucleotide encoding the second free propeptide in the second expression unit to obtain a co-transfected eukaryotic host cell; b) culturing a co-transfected eukaryotic host cell in an appropriate culture medium under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) isolation of FVII or its functional variants from a medium where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17 and 18. 14. A method of obtaining FVII or its functional variants, including a) culturing the eukaryotic host cell according to any one of claims 1 to 3, 7-12 in an appropriate culture medium under conditions that allow the expression of the first polynucleotide and the second polynucleotide; and b) the selection of FVII or its functional variants from a medium where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17 and 18. 15. A method of obtaining FVIIa or its functional variants, including a) transfection of the eukaryotic host cell with the first polynucleotide encoding the first propeptide and FVII or its functional variants in the first expression unit, and transfection with the second polynucleotide encoding the second free propeptide in the second expression unit to obtain a co-transfected eukaryotic host cell; b) culturing in an appropriate culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) the allocation of FVII or its functional variants from the environment and their activation, where each of these first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17 and 18. 16. A method of obtaining FVIIa or its functional variants, including a) culturing the eukaryotic host cell according to any one of claims 1 to 3, 7-18 in the appropriate culture medium under conditions that allow the expression of the first polynucleotide and the second polynucleotide; and b) isolation of FVII or its functional variants from the medium and their activation. 17. The method according to any one of claims 13-16, wherein said first polynucleotide encodes a first human propeptide and FVII. 18. A method of producing vitamin K-dependent proteins, including a) transfection of the eukaryotic host cell with the first polynucleotide encoding the first propeptide and vitamin K-dependent protein in the first expression unit, and transfection with the second polynucleotide encoding the second free propeptide in the second expression unit to obtain a co-transfected eukaryotic host cell; b) culturing in an appropriate culture medium a cotransfected eukaryotic host cell under conditions that allow expression of the first polynucleotide and the second polynucleotide; and c) the selection of vitamin K-dependent proteins from a medium where each of the first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. 19. A method of obtaining vitamin K-dependent proteins, including a) culturing the eukaryotic host cell according to any one of claims 4-12 in an appropriate culture medium under conditions that allow expression of the first polynucleotide and the second polynucleotide; and b) isolation of vitamin K-dependent proteins from the medium. 20. The method according to any one of claims 18 and 19, wherein said first polynucleotide encodes a first propeptide and a vitamin K-dependent protein independently selected from prothrombin, factor IX, FVII, factor X, protein C, protein S, osteocalcin rich in proline protein Gla 1 or matrix protein Gla. 21. The method according to any one of paragraphs.13-20, where the specified first unit of expression is present on the first vector, and the specified second unit of expression is present on the second, separate vector. 22. The method according to any one of claims 13 to 20, wherein said first expression unit and said second expression unit are present on the same vector. 23. The method according to any one of paragraphs.13-22, where the specified culture medium is a medium that does not contain serum. 24. The method according to any one of claims 13-23, wherein said host cell is a mammalian cell. 25. The method according to paragraph 24, where the specified mammalian cell is a human cell. 26. The method of claim 24, wherein said mammalian cell is independently selected from BHK cells, HEK cells, COS cells, or CHO cells. 27. The method according to any one of claims 13-26, wherein said first and second polynucleotides are DNA. 28. The method according to any one of claims 13-26, wherein said first and second polynucleotides are RNA. 29. Recombinant expression vector, where the specified vector contains a polynucleotide encoding a free propeptide in an expression unit, where the specified free propeptide contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18. 30. A recombinant expression vector, wherein said vector comprises a first polynucleotide encoding a first propeptide and FVII or functional variants thereof, in a first expression unit, and a second polynucleotide encoding a second free propeptide, in a second expression unit, where each of said first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 . 31. The recombinant vector of claim 30, wherein said first polynucleotide encodes a human propeptide and FVII. 32. A recombinant expression vector, wherein said vector comprises a first polynucleotide encoding a first propeptide and a vitamin K-dependent protein in a first expression unit, and a second polynucleotide encoding a second free propeptide in a second expression unit, where each of said first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 . 33. The recombinant vector of claim 32, wherein said first polynucleotide encodes a first propeptide and a vitamin K-dependent protein independently selected from prothrombin, factor IX, FVII, factor X, protein C, protein S, osteocalcin enriched in proline Gla 1 or matrix protein Gla. 34. The recombinant vector according to any one of claims 29-33, wherein said first and second polynucleotides are DNA. 35. The recombinant vector according to any one of claims 29-33, wherein said first and second polynucleotides are RNA. 36. A method of obtaining a eukaryotic host cell producing FVII or its analogue, comprising a) transfection of the eukaryotic host cell with the first polynucleotide encoding the first propeptide and FVII or its functional variants in the first expression unit, and b) transfection with a second polynucleotide encoding a second free propeptide in a second expression unit, where each of said first and second propeptides contains an amino acid sequence independently selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18. 37. The method of claim 36, wherein said host cell is further transfected with a polynucleotide encoding gamma glutamyl carboxylase. 38. The method according to any one of claims 36 and 37, wherein said polynucleotides are vectors according to any one of claims 29 to 31, 34 to 35.

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