A method of using eukaryotic expression vectors comprising the bk virus enhancer

Claim 1

A method for producing a functional polypeptide in a eukaryotic host cell wherein a eukaryotic cell transformed with a large DNA virus or the DNA thereof and transformed with a recombinant DNA vector that comprises at least a) a eukaryotic promoter, b) a BK virus enhancer positioned to stimulate the promoter, c) a DNA sequence encoding the functional polypeptide positioned for expression from the promoter, or a eukaryotic cell transformed with a recombinant DNA vector comprising a), b) and c) and d) a DNA sequence encoding an immediate-early gene product of a large DNA virus; is cultured under conditions suitable for expressing c) and d).

Claim 2

A method for producing a functional polypeptide in a eukaryotic host cell wherein a eukaryotic cell transformed with a large DNA virus or the DNA thereof and transformed with a recombinant DNA vector that comprises at least a) a eukaryotic promoter, b) a BK virus enhancer positioned to stimulate the promoter, c) a DNA sequence encoding the functional polypeptide positioned for expression from the promoter, or a eukaryotic cell transformed with a recombinant DNA vector comprising a), b) and c) and d) a DNA sequence encoding an immediate-early gene product of a large DNA virus; is cultured under conditions suitable for expressing c) and d).

Claim 5

The method of 1, 2 or 3 wherein the immediate-early gene product of the large DNA virus is the adenovirus ElA protein, adenovirus E1B protein, pseudorabies virus IE protein, or the herpes simplex virus ICP4 protein.

Claim 6

The method of 1, 2 or 3 wherein the immediate-early gene product of the large DNA virus is the adenovirus ElA protein, adenovirus E1B protein, pseudorabies virus IE protein, or the herpes simplex virus ICP4 protein.

Claim 8

The method of any of Claims 1 to 5 wherein the eukaryotic promoter is an SV40 early promoter, an SV40 late promoter, a BK early promoter, a BK late promoter, a polyoma virus early promoter, a papovavirus early promoter, a polyoma virus late promoter, a papovavirus late promoter, a herpes simplex virus thymidine kinase promoter, an interferon al promoter, a mouse metallothionein promoter, a retrovirus promoter, a β-globin promoter, an adenovirus promoter, the adenovirus-2 late promoter, a sea urchin H2A promoter, a conalbumin promoter, an ovalbumin promoter, a human p globin promoter, the Rous sarcoma virus long terminal repeat promoter.

Claim 10

The method of any of Claims 1 to 7 wherein the DNA that codes for the expression of the immediately- early gene product is contained in the recombinant DNA vector.

Claim 12

The method of any of Claims 1 to 7 wherein the vector comprises the eukaryotic promoter, BK enhancer, and DNA sequence that encodes a useful substance.

Claim 17

The method of any of Claims 10 to 14 wherein the eukaryotic host cell is an adenovirus-transformed, human embryonic kidney cell or a monkey kidney cell.

Claim 29

A method for increasing the activity of a BK enhancer with respect to a eukaryotic promoter which comprises placing the enhancer within 0 to 300 nucleotides upstream of the 5' end of the CAAT region of the eukaryotic promoter, subject to the limitation that the promoter is not the SV40 early promoter.

Claim 30

A method for increasing the activity of a BK enhancer with respect to a eukaryotic promoter which comprises placing the enhancer within 0 to 300 nucleotides upstream of the 5' end of the CAAT region of the eukaryotic promoter, subject to the limitation that the promoter is not the SV40 early promoter.

Claim 36

A method for producing a functional polypeptide in a eukaryotic host cell wherein the cell is transformed with a recombinant DNA vector that contains a eukaryotic promoter and a DNA sequence that encodes the functional polypeptide, the sequence being positioned for expression from the promoter, and wherein the cell containing said vector is cultured under conditions suitable for expression of the functional polypeptide, which comprises: (a) incorporating DNA that encodes the first part of the tripartite leader of an adenovirus into said vector such that, upon transcription, the mRNA produced encodes the funcional polypeptide and, at the 5' end, contains the first part of the tripartite leader; (b) providing the cell containing the vector of step a) with a DNA sequence that codes for the expression of a VA gene product of the adenovirus; and (c) culturing the cell of step b) under conditions suitable for expressing the VA gene product and for stimulating translation of the mRNA, subject to the limitation that the mRNA does not contain the entire tripartite leader of the adenovirus.

Claim 37

A method for producing a functional polypeptide in a eukaryotic host cell wherein the cell is transformed with a recombinant DNA vector that contains a eukaryotic promoter and a DNA sequence that encodes the functional polypeptide, the sequence being positioned for expression from the promoter, and wherein the cell containing said vector is cultured under conditions suitable for expression of the functional polypeptide, which comprises: (a) incorporating DNA that encodes the first part of the tripartite leader of an adenovirus into said vector such that, upon transcription, the mRNA produced encodes the funcional polypeptide and, at the 5' end, contains the first part of the tripartite leader; (b) providing the cell containing the vector of step a) with a DNA sequence that codes for the expression of a VA gene product of the adenovirus; and (c) culturing the cell of step b) under conditions suitable for expressing the VA gene product and for stimulating translation of the mRNA, subject to the limitation that the mRNA does not contain the entire tripartite leader of the adenovirus.

Claim 39

A method for producing a protein that is naturally gamma carboxylated, properly folded, and processed and wherein the protein is encoded in a recombinant DNA vector such that the protein is expressed when a eukaryotic host cell containing the vector is cultured under suitable expression conditions, which comprises: (a) inserting the vector into an adenovirus-transformed, human embryonic kidney cell; and (b) culturing said host cell of step a) under growth conditions and in media containing sufficient vitamin K for carboxylation.

Claim 40

A method for producing a protein that is naturally gamma carboxylated, properly folded, and processed and wherein the protein is encoded in a recombinant DNA vector such that the protein is expressed when a eukaryotic host cell containing the vector is cultured under suitable expression conditions, which comprises: (a) inserting the vector into an adenovirus-transformed, human embryonic kidney cell; and (b) culturing said host cell of step a) under growth conditions and in media containing sufficient vitamin K for carboxylation.