Vectors and methods for recombinant protein expression

Abstract

There is disclosed an expression vector utilizing an internal polyadenylation signal. The internal polyadenylation signal is inserted between a DNA encoding a protein of interest and a DNA encoding a selectable marker, and allows a single promoter to generate both monocistronic messages and dicistronic messages. Similar, multicistronic vectors can also be prepared. Also disclosed are methods of using the expression vector utilizing an internal polyadenylation signal, host cells transfected therewith, stable pools of cells transfected with an expression vector utilizing an internal polyadenylation signal, and clones of such transfected cells.

What is claimed is:  
     
         1 . An expression vector comprising, in the following order, a promoter sequence, a first coding sequence, a polyadenylation site, and a second coding sequence, wherein no promoter sequence occurs between the internal polyadenylation site and the second coding sequence.  
     
     
         2 . The expression vector of  claim 1 , wherein the second coding sequence encodes a selectable marker.  
     
     
         3 . The expression vector of  claim 2 , wherein the selectable marker is dihydrofolate reductase.  
     
     
         4 . The expression vector of  claim 1  wherein the internal polyadenylation site is selected from the group consisting of SV40 late polyadenylation site (SEQ ID NO:1), SV40 early polyadenylation site (SEQ ID NO:3), and a mutant SV40 late polyadenylation site consisting essentially of nucleotides 80 through 222 of SEQ ID NO:1.  
     
     
         5 . The expression vector of  claim 2  wherein the internal polyadenylation site is selected from the group consisting of SV40 late polyadenylation site (SEQ ID NO:1), SV40 early polyadenylation site (SEQ ID NO:3), and a mutant SV40 late polyadenylation site consisting essentially of nucleotides 80 through 222 of SEQ ID NO:1.  
     
     
         6 . The expression vector of  claim 3  wherein the internal polyadenylation site is selected from the group consisting of SV40 late polyadenylation site (SEQ ID NO:1), SV40 early polyadenylation site (SEQ ID NO:3), and a mutant SV40 late polyadenylation site consisting essentially of nucleotides 80 through 222 of SEQ ID NO:1.  
     
     
         7 . The expression vector of  claim 1  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         8 . The expression vector of  claim 2  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         9 . The expression vector of  claim 3  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         10 . The expression vector of  claim 4  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         11 . The expression vector of  claim 5  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         12 . The expression vector of  claim 6  wherein an internal ribosome entry site (IRES) is inserted between the DNA encoding the internal polyadenylation site and the DNA encoding the selectable marker such that the IRES is operably linked to the selectable marker.  
     
     
         13 . A stable pools of cells transfected with an expression vector according to  claim 7 .  
     
     
         14 . A stable pools of cells transfected with an expression vector according to  claim 8 .  
     
     
         15 . A stable pools of cells transfected with an expression vector according to  claim 9 .  
     
     
         16 . A stable pools of cells transfected with an expression vector according to  claim 10 .  
     
     
         17 . A stable pools of cells transfected with an expression vector according to  claim 11 .  
     
     
         18 . A stable pools of cells transfected with an expression vector according to  claim 12 .  
     
     
         19 . A cell line cloned from the pool of  claim 13 .  
     
     
         20 . A cell line cloned from the pool of  claim 14 .  
     
     
         21 . A cell line cloned from the pool of  claim 15 .  
     
     
         22 . A cell line cloned from the pool of  claim 16 .  
     
     
         23 . A cell line cloned from the pool of  claim 17 .  
     
     
         24 . A cell line cloned from the pool of  claim 18 .  
     
     
         25 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 13  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         26 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 14  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         27 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 15  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         28 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 16  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         29 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 17  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         30 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 18  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         31 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 19  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         32 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 20  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         33 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 21  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         34 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 22  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         35 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 23  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         36 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 24  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         37 . The expression vector of  claim 1 , which further comprises a second polyadenylation site following the second coding sequence and operably linked thereto.  
     
     
         38 . An expression vector comprising, in the following order, a promoter sequence, a first coding sequence, a polyadenylation site consisting essentially of nucleotides 80 through 222 of SEQ ID NO:1, and a second coding sequence, wherein no promoter sequence occurs between the internal polyadenylation site and the second coding sequence.  
     
     
         39 . A stable pool of mammalian cells transfected with an expression vector according to  claim 38 .  
     
     
         40 . A cell line cloned from the pool of  claim 39 .  
     
     
         41 . A method for obtaining a recombinant protein, comprising culturing a stable pool of cells according to  claim 39  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         42 . A method for obtaining a recombinant protein, comprising culturing a cell line according to  claim 40  under conditions promoting expression of the protein, and recovering the protein.  
     
     
         43 . A mammalian host cell containing an expression vector according to  claim 7 .  
     
     
         44 . A mammalian host cell containing an expression vector according to  claim 8 .  
     
     
         45 . A mammalian host cell containing an expression vector according to  claim 9 .  
     
     
         46 . A mammalian host cell containing an expression vector according to  claim 10 .  
     
     
         47 . A mammalian host cell containing an expression vector according to  claim 11 .  
     
     
         48 . A mammalian host cell containing an expression vector according to  claim 36 .  
     
     
         49 . The expression vector of  claim 38 , which further comprises a second polyadenylation site following the second coding sequence and operably linked thereto.