Method of using eukaryotic expression vectors comprising the BK virus enhancer

Abstract

The present invention is a method of using the BK enhances in tandem with a eukaryotic promoter to promote transcription of DNA that encodes a useful substance. The method of the present invention requires the presence of the E1A gene produce for maximum expression of the useful substance. The present invention also comprises a number of useful expression vectors that comprise the BK enhancer in tandem with the adenovirus 2 late promoter positioned to drive expression of a variety of proteins, such as protein C, chloramphenicol acetyltransferase, and tissue plasminogen activator. The present invention further comprises a method for increasing the activity of the BK enhancer involving placement of the BK enhancer immediately upstream of the eukaryotic promoter used in tandem with the BK enhancer to drive expression of a useful substance. Furthermore, the present invention also comprises a method for coamplification of genes in primate cells. Additionally, the invention further comprises the recombinant human protein C molecule produced in 293 cells which comprises novel glycosylation patterns.

I claim:  
     
       1. The  A recombinant human protein C molecule produced by inserting a vector comprising the DNA encoding human protein C into an adenovirus-transformed host con then culturing said host cell under growth conditions suitable for production of said recombinant human protein C. 
     
     
       2. The recombinant human protein C molecule of  claim 1  wherein the adenovirus-transformed, host cell is selected from the group consisting of AV12 cells and human embryonic kidney 293 cells. 
     
     
       3. The recombinant human protein C molecule of  claim 2  wherein the adenovirus-transformed host cell is an AV12 cell. 
     
     
       4. The recombinant human protein C molecule of  claim 2  wherein the adenovirus transformed host cell is a human embryonic kidney 293 cell. 
     
     
       5. Human protein C having a glycosylation pattern containing N-acetylgalactosamine (GalNAc). 
     
     
       6. The human protein C of  claim 5 , wherein the protein C is human protein C zymogen. 
     
     
       7. The human protein C of  claim 5 , wherein the protein C is activated human protein C. 
     
     
       8. The human protein C of  claim 5 , wherein said human protein C has at least  2 . 6  moles of N-acetylgalactosamine per mole of protein C. 
     
     
       9. Human protein C produced by introducing DNA encoding protein C into a cell and expressing said protein C in said cell, wherein said protein C has a glycosylation pattern containing N-acetylgalactosamine (GalNAc). 
     
     
       10. The human protein C of  claim 9 , wherein the protein C is human protein C zymogen. 
     
     
       11. The human protein C of  claim 9 , wherein the human protein C is activated protein C produced by introducing DNA encoding protein C into a cell, expressing said protein C in said cell, and activating the protein C. 
     
     
       12. The human protein C of  claim 9 , wherein said cell is an adenovirus-transformed host cell. 
     
     
       13. The human protein C of  claim 10 , wherein said cell is an adenovirus-transformed host cell. 
     
     
       14. The activated human protein C of  claim 11 , wherein said cell is an adenovirus-transformed host cell. 
     
     
       15. The activated human protein C of  claim 14 , wherein the adenovirus-transformed host cell is selected from the group consisting of AV 12  cells and human embryonic kidney  293  cells. 
     
     
       16. The activated human protein C molecule of  claim 14 , wherein the adenovirus-transformed host cell is a human embryonic kidney  293  cell. 
     
     
       17. Human protein C having increased anticoagulant activity as compared to plasma human protein C. 
     
     
       18. A recombinant human protein C molecule of  claim 1 , wherein the human protein C is activated protein C produced by inserting a DNA vector encoding protein C into an adenovirus-transformed host cell, culturing said host cell under conditions suitable for production of said recombinant protein; and activating the protein C to produce activated protein C. 
     
     
       19. The human protein C of  claim 5 , wherein said protein C contains fucose in an amount of at least about  4 . 0  moles fucose per mole of human protein C. 
     
     
       20. The human protein C of  claim 5 , wherein said protein C contains N-acetylgalactosamine in an amount of at least about . 62  moles N-acetylgalactosamine per mole of human protein C. 
     
     
       21. The human protein C of  claim 5 , wherein said protein C contains oligosaccharide chains which are N-linked and does not contain O-linked oligosaccharide chains. 
     
     
       22. The human protein C of  claim 5 , wherein said protein C contains oligosaccharide chains which are N-linked. 
     
     
       23. The human protein C of  claim 5 , wherein said protein C contains oligosaccharide chains which do not contain O-linked oligosaccharide chains. 
     
     
       24. The human protein C of  claim 5 , wherein said protein C is fully γ-carboxylated and glycosylated at positions  97 ,  248 ,  313  and  329 . 
     
     
       25. The human protein C of  claim 5 , wherein said protein C contains less than about  10  moles sialic acid per mole of human protein C. 
     
     
       26. Human protein C which differs from human plasma protein C in that the human protein C has a lower content of sialic acid residues and N-acetylgalactosamine residues are present. 
     
     
       27. The human protein C of  claim 5 , wherein said protein C contains about  4 . 8  moles fucose per mole of human protein C. 
     
     
       28. The human protein C of  claim 5 , wherein said protein C contains about  2 . 6  moles N-acetylgalactosamine per mole of human protein C. 
     
     
       29. The human protein C of  claim 5 , wherein said protein C contains about  12 . 4  moles N-acetylglucosamine per mole of human protein C. 
     
     
       30. The human protein C of  claim 5 , wherein said protein C contains about  6 . 0  moles galactose per mole human protein C. 
     
     
       31. The human protein C of  claim 5 , wherein said protein C contains about  8 . 5  moles mannose per mole human protein C. 
     
     
       32. The human protein C of  claim 5 , wherein said protein C contains about  5 . 4  moles sialic acid per mole human protein C. 
     
     
       33. Human protein C having about  4 . 8  moles fucose per mole of human protein C, about  2 . 6  moles N-acetylgalactosamine per mole of human protein C, about  12 . 4  moles N-acetylglucosamine per mole of human protein C, about  6 . 0  moles galactose per mole human protein C, about  8 . 5  moles mannose per mole human protein C and about  5 . 4  moles sialic acid per mole human protein C.