Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-04-25
2001-12-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
Reexamination Certificate
active
06329176
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for high efficiency release of recombinant proteins in eukaryotic cells or more specifically, for enhancing the secretion of Factor VII by co-expression with Kex2 endoprotease in cultured cells of mammalian origin.
2. Description of the Related Art
Advances in cell culture and recombinant DNA technologies have facilitated the expression of a variety of proteins of therapeutic or other economic value using genetically engineered cells. The expression of many biologically active therapeutic proteins, which are derived from higher eukaryotic sources, often requires specific post-translational modifications which do not naturally occur in lower eukaryotic or prokaryotic cells, thus necessitating the use of cells derived from higher eukaryotic sources. For example, the expression of glycoproteins in mammalian cells has the advantage of providing proteins that contain natural glycosylation. Mammalian-produced glycoproteins contain outer chain carbohydrate moieties which are markedly different from the outer chain carbohydrate moieties present on glycoproteins produced from lower eukaryotes. The use of mammalian cells as hosts for the production of secreted mammalian proteins has the significant advantage over secretion from lower eukaryotes in that mammalian cells have a secretory system that readily recognizes and properly processes secretion-directed proteins, which is not necessarily true for lower eukaryotes.
Efficient expression of coding sequences in eukaryotic hosts may also require the expression of associated proteins that are required for the processing, stabilization or modification of the protein to achieve biological activity. Optimal expression of biologically active recombinant proteins may also be dependent upon the presence of specific translation and/or transcription factors. These proteins may be present in host cells at such low levels that efficient expression of recombinant proteins is limited. Examples of proteins that require specific post-translational modification include certain coagulation factors, which require gamma-carboxylation of specific glutamic acid residues for biological activity and may also require the conversion of specific aspartic acid residues to beta-hydroxy aspartic acid for biological activity.
Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually give rise to a fibrin clot. Generally, the blood components which participate in the so-called coagulation cascade are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytically active enzymes by the action of an activator which in itself is an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as active factors are designated by adding a lower case “a” suffix (e.g. Factor VIIa).
Factor VII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive (Williams et al.,
J. Biol. Chem.
264, 1989, pp. 7536-7543; Rao et al.,
Proc. Natl. Acad. Sci. USA
85, 1988, pp. 6687-6691). Single-chain Factor VII may be converted to two-chain Factor VIIa by Factor Xa, Factor XIIa, Factor IXa or thrombin in vitro. Factor Xa is believed to be a major physiological activator of Factor VII. Like several other plasma proteins involved in hemostasis, Factor VlI depends on vitamin K for expression in an active form, vitamin K being required for the &ggr;-carboxylation of multiple glutamic acid residues clustered in the N-terminus of the protein. The &ggr;-carboxyglutamic acid (Gla)-containing domain is followed by two domains that are homologous to the epidermal growth factor precursor (EGF) whereas the serine protease part occupies the C-terminal half of the molecule.
Proteins involved in the coagulation cascade are also often processed to mature proteins by cleavage at dibasic amino acid residues. Thus, Factor VII is synthesized with an N-terminal 38 amino acids propeptide, which is cleaved off C-terminally to two pairs of arginins (R-R-R-R). There are several candidate enzymes which might be involved in this processing in vivo, some of which operate preferentially in the endoplasmic reticulum (ER) and some in the Golgi apparatus in a membrane bound form.
Another important post-translational modification of Factor VII is gamma-carboxylation of 10 glutamic acid residues located close to the cleavage point of the propeptide. The sequence of events is indicated by the fact that the presence of the propeptide and the correct sequence of it seems important for the process of gamma-carboxylation (Busby et al., Curr. Adv. in Vit. K. Res. 173-181 (1987) and Ul-rich et al., J. Biol. Chem. 263 (20) 9697-9702 (1988)), which occurs in the ER catalyzed by a membrane-bound carboxylase.
Kex2 endoprotease of Saccharomyces yeast is a protease that specifically processes the precursor of mating type &agr;-factor and a killer factor. The properties of the Kex2 endoprotease are reported to be the following: (1) Kex2 cleaves at the C-terminal of Lys-Arg sequence for excision of mating type &agr;-factor from its precursor, and at the C-terminal of Lys-Arg sequence and Pro-Arg sequence to release mature killer factor; (2) a purification thereof was attempted, and it was found that the enzyme is present in a membrane fraction and requires calcium ions for activation thereof; (3) Kex2 is a glycoprotein having a molecular weight of 100 to 120 K Dalton; (4) Kex2 specifically cleaves at the C-terminal of sequences Arg-Arg, Lys-Arg, and Pro-Arg (BBRC, 144, 807-814, 1987).
In WO 90/01550 plasmids are disclosed carrying polycistronic expression units including an intercistronic leader. In U.S. Pat. No. 5,460,950 host cells are disclosed expressing PACE, a human endoprotease, capable of cleaving precursor polypeptides where the PACE encoding sequence and the precursor polypeptide encoding sequence is operably linked to an expression control sequence permitting co-expression of the two sequences.
SUMMARY OF THE INVENTION
The present invention is related to a method for producing Factor VII comprising
a) cultivating a mammalian cell line comprising a DNA sequence encoding a yeast endoprotease or a derivative thereof and a DNA sequence encoding FVII in a suitable culture medium; and
b) isolating Factor VII from the medium.
According to one embodiment of the present invention, the yeast endoprotease is preferably derived from a Saccharomyces strain and is preferably a Kex2 like endoprotease. The yeast endoprotease may be truncated at its C-terminal end thereby being deprived of its transmembrane region. Further, an ER retention signal may be added to the C-terminal end of the truncated endoprotease. The yeast endoprotease is preferably the Kex2 endoprotease from
Saccharomyces cerevisiae.
The culture medium is preferably a serum free medium and the mammalian cell line is preferably a CHO cell line or a BHK cell line.
REFERENCES:
patent: 5460950 (1995-10-01), Barr et al.
patent: 5516650 (1996-05-01), Foster et al.
patent: 0 319 944 (1989-06-01), None
patent: 0 548 012 B1 (1997-03-01), None
Hagen et al. Proc. Natl. Acad. Sci. USA, 83:2412-2416, 1986.*
Mather. in Goeddel et al., Gene Expression Technology, Methods in Enzymology vol. 185, 1990, p. 576.
Nielsen Lars Søegaard
Wiberg Finn
Woldike Helle
Gansheroff Lisa
Green, Esq. Reza
Ketter James
Novo Nordisk A S
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