Expression vector, methods for the production of heterologous gene products and for the selection of recombinant cells producing high levels of such of such products

Abstract

An expression vector for eukaryotic cells comprising a gene which codes for a protein of interest, functionally linked to a hamster-ubiquitin/S27a-promoter and a gene which codes for a fluorescent protein. Preferably the expression vector also contains an amplifiable selectable marker gene. The invention also describes host cells, preferably mammalian cells, which have been transfected with the expression vector, processes for producing heterologous gene products and a method of selecting high-producing cells.

1 . An expression vector comprising: (a) a gene which codes for a protein/product of interest, functionally linked to a hamster-ubiquitin/S27a-promoter; and (b) a gene which codes for a fluorescent protein. 
     
     
         2 . The expression vector according to  claim 1 , comprising an amplifiable selectable marker gene. 
     
     
         3 . The expression vector according to  claim 1 , comprising one or more enhancers functionally linked to the promoter. 
     
     
         4 . The expression vector according to  claim 1 , further comprising an internal ribosomal entry site (IRES) which allows bicistronic expression of the gene which codes for the fluorescent protein, and of the gene which codes for the protein/product of interest. 
     
     
         5 . The expression vector according to  claim 2 , wherein the gene which codes for the fluorescent protein and the amplifiable selectable marker gene are located in one or in two separate transcription units. 
     
     
         6 . The expression vector according to  claim 1 , wherein the functional linking does not take place via intron sequences. 
     
     
         7 . The expression vector according to  claim 1 , wherein the amplifiable selectable marker gene codes for dihydrofolate-reductase (DHFR) or a fusion protein of the fluorescent protein and DHFR. 
     
     
         8 . The expression vector according to  claim 3 , wherein the enhancer is a CMV or SV40 enhancer. 
     
     
         9 . The expression vector according to  claim 1 , further comprising at least one polyadenylation signal. 
     
     
         10 . An expression vector according comprising a multiple cloning site for the incorporation of a gene which codes for a protein/product of interest. 
     
     
         11 . A eukaryotic host cell transfected with an expression vector according to  claim 2 . 
     
     
         12 . A host cell according to  claim 11 , which is a mammalian cell. 
     
     
         13 . A host cell according to  claim 11 , which is a CHO cell. 
     
     
         14 . The host cell according to  claim 11 , additionally transfected with one or more vectors comprising one or more genes encoding one or more other proteins/products of interest and at least one other selectable marker. 
     
     
         15 . A process for preparing a heterologous gene product, comprising cultivating a host cell according to  claim 11  under conditions which allow expression of the gene product, and isolating the gene product from the culture or culture medium. 
     
     
         16 . A process for preparing a heteromeric protein/product, comprising co-transfecting the host cell according to  claim 14  with expression vectors which code for different subunits of the heteromeric protein/product under conditions which allow expression of the heteromeric protein/product, and isolating the heteromeric protein/product from the culture or culture medium. 
     
     
         17 . The process according to  claim 16 , wherein the heteromeric protein is an antibody. 
     
     
         18 . The process according to  claim 15 , further comprising subjecting the host cell to one or more gene amplification steps in the presence of an amplifying agent. 
     
     
         19 . The process according to  claim 18 , wherein the amplifiable selectable marker is dihydrofolate reductase (DHFR) and the amplifying agent is methotrexate. 
     
     
         20 . The process according to  claim 18 , wherein the host cell is subjected to only one gene amplification step with methotrexate. 
     
     
         21 . The process according to  claim 15 , wherein the host cell is cultured in a serum-free culture medium. 
     
     
         22 . The process according to  claim 15 , wherein the host cell is cultivated in suspension culture. 
     
     
         23 - 26 . (canceled)