Method of using eukaryotic expression vectors comprising the BK virus enhancer

Abstract

The present invention is a method of using the BK enhancer 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 product 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. In a method for producing a protein that is naturally  γ-carboxylated, properly folded and processed and wherein said protein is encoded in a recombinant DNA vector such that said protein is expressed when a eukaryotic host cell containing said vector is cultured under suitable expression conditions, the improvement comprising: 
       (a) inserting said vector into an adenovirus-transformed host cell; and  
       (b) culturing said host cell of step a) under growth conditions and in media containing sufficient vitamin K for carboxylation.  
     
     
       2. The method of  claim 1  wherein the adenovirus-transformed host cell is selected from the group consisting of the human embryonic kidney 293 and the AV12 cell lines. 
     
     
       3. The method of  claim 2 , wherein said γ-carboxylated protein is human protein C. 
     
     
       4. The method of  claim 3 , wherein said host cell cultured in step (b) is selected from the group consisting of 293/pLPC, 293/pLPC4, 293/pLPChyg1, 293/pLPCdhfr1, 293/pLPChd1, 293/pLPChT1, 293/p4-14, AV12/pLPC, AV12/pLPC4, AV12/pLPChyg1, AV12/pLPCdhfr1, AV12/pLPChd1, AV12/pLPChT1, and AV12/p4-14 host cells. 
     
     
       5. The method of  claim 1 , wherein said vector comprises a BK virus enhancer.  
     
     
       6. The method of  claim 5 , wherein said host cell expresses an immediate- early gene product of a DNA virus.    
     
     
       7. The method of  claim 6 , wherein said immediate- early gene product of a DNA virus is an EIA gene product of adenovirus.    
     
     
       8. The method of  claim 5 , wherein said BK virus enhancer is present in tandem with a eukaryotic promoter.  
     
     
       9. The method of  claim 8 , wherein said eukaryotic promoter is an adenovirus late promoter ( MLP ).  
     
     
       10. The method of  claim 1 , wherein said vector comprises a BK enhancer- adenovirus late promoter cassette.    
     
     
       11. The method of  claim 2 , wherein the adenovirus- transformed host cell is a human embryonic kidney  293  cell having DNA encoding the murine dihydrofolatereductase gene, the nascent human protein C gene, the SV 40  early promoter, the BK enhancer and the adenovirus  2  late promoter, wherein said SV 40  early promoter and said adenovirus  2  late promoter are used in tandem.    
     
     
       12. The method of  claim 3 , further comprising activating the expressed human protein C.