Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Lyase
Patent
1987-10-27
1992-03-31
Robinson, Douglas W.
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Lyase
C12N 988
Patent
active
051007983
DESCRIPTION:
BRIEF SUMMARY
The invention relates to a method for the extraction of a vitamin-K-dependent carboxylase complex from biological material which can be used for the conversion of, for example, blood coagulation factor IX precursors prepared via recombinant DNA technology, into the blood coagulation factor IX.
As is known, patients who lack the blood coagulation factor IX suffer from haemophilia B. As a possibility of helping these patients concentrates of these coagulation factors are nowadays made on a large scale, which concentrates are extracted from human blood plasma. However, many kinds of difficulty are associated with the injection of human blood products in patients. As difficulties, mention may be made of:
1) the transmission of diseases such as hepatitis and AIDS;
2) the necessity of mounting extensive blood plasma campaigns for the collection of the necessary blood; and
3) the extraction of the respective coagulation factors from human blood plasma is cumbersome and costly.
From Proc. Natl. Acad. Sci. USA, vol. 79, pages 6461-6464, November 1982 and Biochemistry 1983, 22, pages 2087-2097, the possibility is known of isolating and incorporating in bacteria the DNA fragment that codes for factor IX. The said bacteria multiply rapidly and produce at the same time the desired coagulation factor IX, which can be extracted from the culture medium (see Biochemistry 1984, 23, pages 1626-1634). Also eukaryotic cell lines producing recombinant factor IX-like material have been described (Nature 316, 1985, pages 268-270 and pages 271-273, J. Biol. Chem. 261, 1986, pages 9622-9628).
With the above named method the risk of disease transmission is eliminated, there is no dependence on blood donors and, in addition, the production process is cheaper. However, there is still a fundamental problem relating to the coagulation factor IX prepared in this manner. In particular, it is of essential importance for the biological activity of the said factor that it undergoes a so-called post-translational modification. This modification consists of the gamma-carboxylation of the 12 N-terminal glutamic acid residues. As is evident, for example, from Haematologia 18 (2), pages 71-97 (1985), the enzyme which is responsible for this carboxylation reaction is the vitamin-K-dependent carboxylase, which enzyme does not occur in bacteria. Also in eukaryotic cell lines (which do contain carboxylase) the recombinant factor IX is carboxylated for only 1-3% (J. Biol. Chem. 261, 1986, pages 9622-9628). The factor IX referred to above and produced with the aid of DNA recombinant technology is therefore biologically inactive and cannot be used for making up the deficiency in haemophilia B patients. The same problem arises with the uncarboxylated blood coagulation factors II, VII and X produced with the aid of bacteria modified by DNA recombinant technology.
The invention therefore has the object of developing a method of converting uncarboxylated coagulation factors II, VII, X and in particular IX, into the biologically active form. From Biochimica et Biophysica Acta, 714 (1982), pages 361-365 it is known that in principle the starting point for the extraction of the carboxylase complex may be two types of liver, preferably bovine liver, namely: (a) normal liver and (b) liver from cows which have previously been treated with vitamin K antagonists. As a result of this treatment uncarboxylated blood coagulation factors acumulate in the liver, where they are complexed with the carboxylase complex. In the case of the normal liver a "substrate-free" enzyme is thus obtained and in the case of animals treated with vitamin K antagonists an enzyme/substrate complex is obtained. This latter complex is very tight and, as far as is known, can only be split by boiling in 10% SDS.
Although the carboxylase is detectable in liver homogenates and in the microsomal fraction therefrom, preparations of this type are too impure to be used for the carboxylation of, for example, the blood coagulation factor IX precursor. Moreover, a so-called "solid-phase" carboxylase is
REFERENCES:
Girardot, "Vitamin K-Dependent Carboxylase: Partial Purification and Properties of the Enzyme-Substrate Complex", J. Biol. Chem. 257(24), 15008-15011, 1982.
"Vitamin K-Dependent Carboxylase: Evidence for Cofractionation of Carboxylase and Epoxidase Activities, and for Carboxylation of a High-Molecular-Weight Microsomal Protein", Chemical Abstracts, vol. 96, No. 17, Apr. 26, 1982, Summary No. 138618w, Wallin et al., p. 370.
"Vitamin K-Dependent Carboxylation: Synthesis and Biological Properties of Diastereomeric Gamma-Substituted Glutamic Acid Containing Peptidic Substrates", Biological Abstracts, vol. 79, No. 6, 1985, Summary No. 46413, Bory.
"Vitamin K-Dependent Carboxylase: Partial Purification and Properties of the Enzyme-Substrate Complex", Biological Abstracts, vol. 75, No. 11, 1983, Summary No. 79838, Girardot.
"Isolation and Characterization of a cDNA Coding for Human Factor IX", Proc. Natl. Acad. Sci. USA, Biochemistry, vol. 79, Nov. 1982, Kurachi et al., pp. 6461-6464.
"Characterization of the Complementary Deoxyribonucleic Acid and Gene Coding for Human Prothrombin", Biochemistry, vol. 22, No. 9, 1983, Degen et al., pp. 2087-2097.
"Characterization of Bovine Prothrombin mRNA and Its Translation Product", Biochemistry, vol. 23, No. 8, 1984, MacGillivray et al., pp. 1626-1634.
"Vitamin K-Dependent Carboxylase", Haematologia, vol. 18, No. 2, 1985, Vermeer et al., pp. 71-97.
"A Comparison Between Vitamin K-Dependent Carboxylase from Normal and Warfarin-Treated Cows", Biochimica et Biophysica Acta, 1982, Vermeer et al., pp. 361-365.
"Partial Purification of Bovine Liver Vitamin K-Dependent Carboxylase by Immunospecific Adsorption onto Antifactor X", FEBS Letters, vol. 123, No. 2, Jan. 1981, De Metz, pp. 215-218.
"Vitamin K-Dependent Carboxylase: The Carboxylation of Exogenous Substrates in Different Systems", Biochimica et Biophysica Acta, 1985, de Boer-van den Berg et al., pp. 1-5.
Hubbard et al., Proc. Natl. Acad. Sci. USA, vol. 86, Sep. 1989, pp. 6893-6897.
Soute et al., Thrombosis and Haemostasis, vol. 61, 1989, pp. 238-242.
Soute Berry A. M.
Vermeer Cornelis
Rijksuniversiteit Limburg
Robinson Douglas W.
Witz Jean C.
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