Compositions and Methods Relating to Proteins Requiring Gamma-Carboxylation

Abstract

The present invention relates a host cell comprising an expression vector comprising a nucleic acid molecule encoding a protein requiring gamma-carboxylation and associated expression control sequences and a nucleic acid molecule encoding a vitamin K epoxido reductase and associated expression control sequences and a nucleic acid molecule encoding a γ-glutamyl carboxylase and associated control sequences. The invention further relates to a method of producing a protein requiring gamma-carboxylation in high yields.

1 .- 32 . (canceled) 
     
     
         33 . An in vitro host cell comprising:
 a first DNA comprising a sequence encoding factor VII operably linked to a first expression control sequence;   a recombinant second DNA comprising a sequence encoding a vitamin K epoxidoreductase (VKOR) operably linked to a second expression control sequence; and   a third DNA comprising a sequence encoding a γ-glutamyl carboxylase operably linked to a third expression control sequence, wherein mRNA encoding the factor VII and mRNA encoding the VKOR are expressed in the cell in a ratio of at least 10:1.   
     
     
         34 . The in vitro host cell of  claim 33 , wherein mRNA encoding factor VII and mRNA encoding the γ-glutamyl carboxylase are expressed in the cell in a ratio of at least 10:1. 
     
     
         35 . The in vitro host cell of  claim 33 , wherein the first DNA and the second DNA are located on a single expression vector in the cell. 
     
     
         36 . The in vitro host cell of  claim 33 , wherein the first DNA, the second DNA, and the third DNA are located on a single expression vector in the cell. 
     
     
         37 . The in vitro host cell of  claim 33 , wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, and the activity of the first promoter is greater than the activity of the second promoter in the host cell. 
     
     
         38 . The in vitro host cell of  claim 37 , wherein the first promoter is selected from the group consisting of: human cytomegalovirus (hCMV) immediate-early promoter, human elongation factor-1αsubunit gene promoter (eEF-1α), Rous sarcoma virus promoter (pRSV), and human ubiquitin promoter (pUbC). 
     
     
         39 . The in vitro host cell of  claim 38 , wherein the first promoter is hCMV immediate-early promoter, and the second promoter is simian virus 40 (SV40) early promoter. 
     
     
         40 . The in vitro host cell of  claim 37 , wherein the third expression control sequence comprises a third promoter, and the activity of the first promoter is greater than the activity of the third promoter in the host cell. 
     
     
         41 . The in vitro host cell of  claim 33 , wherein the host cell is a mammalian cell. 
     
     
         42 . The in vitro host cell of  claim 33 , wherein the host cell is a yeast cell or an insect cell. 
     
     
         43 . The host cell of  claim 33 , wherein the cell is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, a mouse myeloma (NS0) cell, a human retinal (Per C.6) cell, or an African green monkey fibroblast-like kidney (COS) cell. 
     
     
         44 . A method for producing a composition, the method comprising:
 (a) providing a recombinant cell comprising a first DNA encoding factor VII operably linked to a first expression control sequence, a recombinant second DNA encoding a vitamin K epoxidoreductase (VKOR) operably linked to a second expression control sequence, and a third DNA encoding a γ-glutamyl carboxylase operably linked to a third expression control sequence;   (b) culturing the cell in vitro under conditions suitable for expressing each DNA, wherein (i) mRNA encoding factor VII and mRNA encoding the VKOR are expressed in the cell in a ratio of at least 10:1, and (ii) the factor VII is carboxylated in the cell, thereby producing γ-carboxylated factor VII; and   (c) isolating the γ-carboxylated factor VII or an activated form thereof.   
     
     
         45 . The method of  claim 44 , further comprising:
 (d) preparing a pharmaceutical composition comprising the isolated γ-carboxylated factor VII or an activated form thereof.   
     
     
         46 . The method of  claim 44 , wherein mRNA encoding factor VII and mRNA encoding the γ-glutamyl carboxylase are expressed in the cell in a ratio of at least 10:1. 
     
     
         47 . The method of  claim 44 , wherein both the first and third DNA are located on a single expression vector in the cell. 
     
     
         48 . The method of  claim 44 , wherein the first, second, and third DNA are located on a single expression vector in the cell. 
     
     
         49 . The method of  claim 44 , wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, and the activity of the first promoter is greater than the activity of the second promoter in the cell. 
     
     
         50 . The method of  claim 44 , wherein the first promoter is selected from the group consisting of: human cytomegalovirus (hCMV) immediate-early promoter, human elongation factor-1α subunit gene promoter (eEF-1α), Rous sarcoma virus promoter (pRSV), and human ubiquitin promoter (pUbC). 
     
     
         51 . The method of  claim 50 , wherein the first promoter is hCMV immediate-early promoter and the second promoter is simian virus 40 (SV40) early promoter. 
     
     
         52 . The method of  claim 44 , wherein the first expression control sequence comprises a first promoter, the second expression control sequence comprises a second promoter, the third expression control sequence comprises a third promoter, and the activity of the first promoter is greater than the activity of the third promoter in the cell. 
     
     
         53 . The method of  claim 52 , wherein the first promoter is selected from the group consisting of: hCMV immediate-early promoter, pEF-1α, pRSV, and pUbC. 
     
     
         54 . The method of  claim 52 , wherein the first promoter is hCMV immediate-early promoter, and the third promoter is SV40 early promoter. 
     
     
         55 . The method of  claim 52 , wherein the activity of the first promoter is greater than the activity of each of the second and third promoters in the cell. 
     
     
         56 . The method of  claim 55 , wherein the first promoter is selected from the group consisting of: hCMV immediate-early promoter, pEF-1α, pRSV, and pUbC. 
     
     
         57 . The method of  claim 55 , wherein the first promoter is hCMV immediate-early promoter, and each of the second and third promoters is SV40 early promoter. 
     
     
         58 . The method of  claim 44 , wherein the cell is a mammalian cell. 
     
     
         59 . The method of  claim 44 , wherein the cell is a yeast cell or an insect cell. 
     
     
         60 . The method of  claim 44 , wherein the cell is a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, a mouse myeloma (NS0) cell, a human retinal (Per C.6) cell, or an African green monkey fibroblast-like kidney (COS) cell.