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
1996-11-22
2001-04-03
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S068100, C435S252300, C435S252330, C435S320100, C435S471000, C536S023700
Reexamination Certificate
active
06210929
ABSTRACT:
The invention relates to a new fusion protein, derived from furin or a furin analog, as well as to a method of preparing proteins from proproteins by means of the fusion protein, in particular of von Willebrand factor from pro-von Willebrand factor.
In addition to PACE4, PC1/PC3, PC2, PC4 and PC5/PC6, furin, also termed PACE, belongs to the group of the subtilisin-like serine proteases, which play an important role in the cleavage of proproteins, especially in the secretory synthesis (Van de Ven et al., Crit. Rev. Oncogen., 4:115-136, 1993). Proproteins are post-translationally, intracellularily processed to their mature form by the endogenous protease in the Golgi apparatus. The protease cleavage site comprises a recognition sequence which is characterized by the amino acid sequence Arg-X-Lys/Arg-Arg. The protease furin cleaves proproteins specifically after this consensus sequence (Hosaka et al., J. Biol. Chem. 266:12127-12130, 1991).
The DNA and amino acid sequence of human and murine furin, as well as further proteins with subtilisin-like protease function have been clarified (Roebroek et al., Mol. Biol. Rep. 11: 117-125, 1986, Roebroek et al., EMBO J. 5:2197-2202, 1986, Barr et al., DNA Cell Biol. 10:319-328, 1991, Van den Ouweland et al., Nucleic Acids Res. 17:7101-7102, 1989, Van den Ouweland et al., Nucleic Acids Res. 18:664, 1990, Smeekens et al., 1990, J. Biol. Chem. 265:2997-3000; Smeekens et al., 1991, Proc. Natl. Acad. Sci. USA. 88; 340-344; Kiefer et al., 1991, DNA Cell Bio. 10: 757; Nakayama et al., 1992, J. Bio. Chem. 267:5897-5900, Hatsuzawa et al., 1990. J. Biol. Chem. 265: 22075-22078). The human fur-gene encodes a protein consisting of 794 amino acids, certain functions being allocatable to individual characteristic regions: a catalytic center, a middle domain, a cystein-rich region, a transmembrane and a cytoplasmatic domain (Van de Ven et al., Crit. Rev. Oncogen., 4:115-136, 1993).
Intact furin is incorporated into the membrane system of the Golgi apparatus and there it is functionally active (Bresnahan et al., J. Cell Biol. 111:2851-2859, 1990). A truncated form of the over-expressed native furin of 75-80 kD could be detected in the cell supernatant as secreted protein (Wise et al., Proc. Natl. Acad. Sci. USA 87: 9378-9382, 1990). This naturally secreted furin is known as “shed furin” (Vidricaire et al., Biochem. Biophys. Res. Comm. 195:1011-1018, 1993) and is cleaved N-terminally of the transmembrane portion (Vey et al., J. Cell Biol. 127:1829-1842, 1994).
Furin truncated by genetical engineering, in which the encoding part of the transmembrane and cytoplasmatic domains has been deleted, can also be expressed and secreted correspondingly. Such N-terminal deletions have been described for amino acids &Dgr;714-794 (Leduc et al., J. Biol. Chem. 267:14304-14308, 1992, Molloy et al., J. Biol. Chem. 267:16396-16402, 1992) and for amino acids &Dgr;716-794 (“Sol-PACE”) (Wasley et al., 1993. J. Biol. Chem. 268:8458-8465, Rehemtulla et al., Blood 79:2349-2355, 1992) and for amino acids &Dgr;705-794 (Hatsuzawa et al., 1992. J. Biol. Chem. 267:16094-16099).
Furin mutants additionally comprising a deletion of the cystein-rich region have also been described (Hatsuzawa et al., 1992. J. Biochem. 101:296-301, Creemers et al., 1993. J. Biol. Chem. 268:21826-21834).
The endoproteolytic activity of furin and its selectivity for basic amino acids has first been determined in experiments with pro-von Willebrand factor (pro-vWF). Pro-vWF consists of a propolypeptide with 741 amino acids and mature von Willebrand factor (vWF) with 2050 amino acids (Verweij et al., EMBO J. 5:1839-1847, 1986). The liberation of mature vWF from pro-vWF results from a proteolytic cleavage after Arg763. Transfection of pro-vWF cDNA in eukaryotic expression vectors results in the production of equimolar amounts of the 360 kD pro-vWF and of the 260 kD mature vWF in the cell culture supernatant. vWF is probably processed into its mature form in transfected cells, by endogenously occurring furin (Wise et al., Proc. Natl. Acad. Sci. USA 87:9378-9382, 1990, Van de Ven et al., Mol. Biol. Rep. 14:265-275, 1990).
Among the further proproteins which are cleaved by furin or by subtilisin-like enzymes, respectively, are a series of hormones and growth factors (e.g., proactivin A, hepatocyte-growth factor), plasma proteins (albumin, factor VII, factor IX, factor X), receptors (insulin pro-receptor), viral proteins (e.g. HIV-1 gp160, influenza virus haemagglutinin) as well as bacterial proteins (diphteria toxin, anthrax toxin) (Decroly et al., J. Biol. Chem. 269:12240-12247, 1994, Stieneke-Gröber et al., EMBO J. 11:2407-2414, 1992, Barr, Cell 66:1-3, 1991, Wasley et al., J. Biol. Chem. 268:8458-8465, 1993, Klimpel et al., Proc. Natl. Acad. Sci. USA 89:10277-10281, 1992, Tsuneoka et al., J. Biol. Chem. 268:26461-26465, 1993, Bresnahan et al., J. Cell. Biol. 111:2851-2859, 1990, Hosaka et al., J. Biol. Chem. 266:12127-12130, 1991, Vey et al., J. Cell. Biol. 127:1829-1842, 1994).
By co-expression of the nucleic acid sequences encoding intact furin and a proprotein in eukaryotic cell cultures, an increased processing of the proproteins has been achieved in vivo. This has been demonstrated, e.g., for pro-factor IX (Wasley et al., J. Biol. Chem. 268:8458-8465, 1993) and for pro-vWF (WO 91/06314, Van de Ven et al., Mol. Bio. Rep. 14:265-275, 1990, Rehemtulla et al., Blood 79:2349-2355, 1992).
Beside the co-expression of intact furin with proproteins, there have also been attempts to express truncated furin together with proproteins. Deleted furin is enzymatically active when co-expressed in vivo and is secreted; the enzymatic activity of such deletion mutants could be detected inter alia in the processing of pro-factor IX (Wasley et al., J. Biol. Chem. 268:8458-8465, 1993) and of pro-vWF (Rehemtulla et al., Blood 79: 2349-2355, 1992). Co-expression experiments with furin deletion mutants have shown that the transmembrane and the cytoplasmatic part of the protein are not essential to the catalytic function (Rehemtulla et al., Proc. Natl. Acad. Sci. USA 89: 8235-8239, 1992).
WO 91/06314 discloses the recombinant expression of furin in prokaryotic and eukaryotic cells, the preparation of furin fusion proteins, deletion mutants and fragments, the purification of recombinantly prepared furin, as well as the possible use of purified furin for the processing of proproteins in vitro in general.
WO 92/09698 describes the expression of PACE (furin), the co-expression with inactive precursors of proteins, such as, e.g., pro-vWF, as well as the preparation of fusion proteins. To enrich PACE it has been suggested therein to isolate PACE capable of secretion, by conventional methods.
Stieneke-Gröber et al. (EMBO J. 11:2407-2414, 1992) describe the in vitro cleavage of influenza virus HA protein by means of purified furin. Decroly et al. (J. Biol. Chem. 269:12240-12247, 1994) describe the in vitro cleavage of HIV gp160 by means of furin.
In experiments with C-terminally shortened furin, the cleavage of pro-albumin and complement Pro-C3 (Oda et al., Biochem. Biophys. Res. Commun. 189:1353-1361, 1992), anthrax toxin (Klimpel et al., Proc. Nastl. Acad. Sci. USA 89:10277-10281, 1992), diphtheria toxin (Tsuneoka et al., J. Biol. Chem. 268: 26461-26465, 1993) and pro-factor IX (Wasley et al., J. Biol. Chem. 268:8458-8468, 1993, Bristol et al., Biochemistry 33:14136-14143, 1994) has been carried out successfully in vitro.
So far, it has not been possible to demonstrate in vitro processing of pro-vWF by means of furin. Rehemtulla et al. (Blood 79:2349-2355, 1992, and Proc. Natl. Acad. Sci. USA 89:8235-8239, 1992) describe that by mixing supernatants of cells transfected with pro-vWF and deleted furin (“PACE SOL”), respectively, pro-vWF is not processed to vWF. Contrary therto, both synthetic substrates (Rehemtulla et al., Proc. Natl. Acad. Sci. USA 89:8235-8239, 1992) and also pro-factor IX (Bristol et al., Biochemistry 33:14136-14143, 1994) could be cleaved in vitro by means of purified “PACE SOL”. For pro-vWF it has, furthermore, repeate
Dorner Friedrich
Eibl Johann
Falkner Falko-Guenter
Fischer Bernhard
Schlokat Uwe
Achutamurthy Ponnathapu
Baxter Aktiengesellschaft
Foley & Lardner
Moore William W.
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