Vitamin K is a cofactor of γ-glutamyl carboxylase (GGCX) which has a role in post-translational modification of various types of vitamin K-dependent proteins. Predetermined glutamic residues in various vitamin K-dependent proteins are carboxylated by GGCX in the presence of vitamin K, resulting in conversion into γ-carboxyglutamic acid (Gla). The γ-glutamyl carboxylation is known to be very important for biological functions of the vitamin K-dependent proteins (for example, blood coagulation, bone metabolism, and signal transduction). For example, the blood coagulation factor II (prothrombin) which is a kind of the vitamin K-dependent proteins or the factor X can be bound to phospholipids of the cell membrane (the site for the coagulation reaction) by γ-glutamyl carboxylation. As a result, the activation responses from other factors are received.
Vitamin K-dependent blood coagulation factor such as the factor II or factor X is mainly prepared by using the plasma from human or bovine as a raw material. However, the incorporation of infectious materials into the raw material or a difference between the production lots causes a problem. Therefore, methods for producing a vitamin K-dependent blood coagulation factor by the recombinant DNA technique using a mammalian cell have been recently studied and developed.
However, it is known that the vitamin K-dependent blood coagulation factor obtained by the expression system using a mammalian cell is not completely in the γ-glutamyl carboxylated form. It is generally difficult to activate the coagulation factor which is not in the γ-glutamyl carboxylated form. Accordingly, it is desirable that the recombinant vitamin K-dependent blood coagulation factor is sufficiently in the γ-glutamyl carboxylated form when obtained in the protein expression system, from the viewpoint of industrial application. Therefore, a method for producing a γ-glutamyl carboxylated protein, comprising co-expressing a vitamin K-dependent protein and GGCX in the expression system using a mammalian cell has been developed (refer to US Pub 2005/164367).
On the other hand, in addition to GGCX, vitamin K epoxide reductase (VKOR) and DT-diaphorase (also referred to as “NAD(P)H-dependent quinone oxidoreductase 1”; NOQ1) are known to be involved in the γ-glutamyl carboxylation of the vitamin K-dependent protein (refer to Tie J-K. et al., Blood. vol. 117, and p. 2967-2974 (2011)). Here, the enzyme which is directly involved in the γ-glutamyl carboxylation is GGCX, while VKOR and NQO1 are enzymes which are involved in the recycling of vitamin K. In recent years, a method for producing a γ-glutamyl carboxylated protein, comprising co-expressing a vitamin K-dependent protein, GGCX, and VKOR in the expression system using a mammalian cell has been developed (refer to US Pub 2009/100533).