Impaired dominant selectable marker sequence and intronic insertion strategies for enhancement of expression of gene product and expression vector systems comprising same

Abstract

Disclosed herein are fully impaired consensus Kozak sequences which are most typically used with dominant selectable markers of transcriptional cassettes which are a part of an expression vector, These vectors are most typically utilized in the expression of proteins in mammalian expression systems. As defined, disclosed and claimed herein, a “fully impaired consensus Kozak” comprises the following sequence: −3        +1  Pyxx ATG Pyxx where: “x” is a nucleotide selected from the group consisting of adenine (A), quanine (G), cytosine (C) or thymine (T)/uracil (U); “Py” is a pyrimidine nucleotide, ie C or T/U; “ATG” is a codon encoding for the amino add methionine, the so-called “start” codon; and −3 and +1 are directional reference points vis-a-vis ATG, ie −3 is meant to indicate three nudeotides upstream of ATG and +1 is meant to indicate one nucleotide downstream of ATG. Dominant selectable markers further comprising artificial intronic insertion regions are further disclosed.

What is claimed is:  
     
         1 . An expression vector for expressing a protein of interest by recombinant deoxyribonucleic acid techniques, raid vector comprising at least one dominant selectable marker, wherein the translation initiation start site of said marker comprises the following sequence:  
       
         
           
                 
                 
               
                     
                     
                 
                     
                   −3       +1 
                 
                     
                   Pyxx ATG Pyxx 
                 
                     
                     
                 
             
                
                
                
                
               
            
           
         
       
       where “Py” is a pyrimidine nucleotide; “x” is a nucleotide; and the numerical designations are relative to the codon “ATG”.  
     
     
         2 . The expression vector of  claim 1  wherein the nucleic acid sequence encoding for the protein of interest is co-linked to said dominant selectable marker.  
     
     
         3 . The expression vector of  claim 1  wherein said dominant selectable marker is selected from the group consisting of: herpes simplex virus thymidine kinase, adenosine deamixiase, asparagine synthetase, Salmonella his D gene, xanthine guanine phosphoribosyl transferase, hygromycin B phosphotransferase, and neomycin phosphotransferase.  
     
     
         4 . The expression vector of  claim 1  wherein said translation initiation start site sequence is selected from the group consisting of TxxATGCxx; CxxATGCxx; CxxATGTxx; and TxxATGTxx, where “x” is a nucleotide, with the proviso that the codon “Txx” downstream of the ATG codon does not encode a stop codon.  
     
     
         5 . The expression vector of  claim 1  wherein said translation initiation start site sequence is TxxATGCxx, where “x” is a nucleotide.  
     
     
         6 . The expression vector of  claim 1  wherein said translation initiation start site sequence is TCCATGCT.  
     
     
         7 . The expression vector of  claim 1  wherein said translation initiation start site sequence is located within a secondary structure.  
     
     
         8 . The expression vector of  claim 1  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 1000 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 1000 nucleotides.  
     
     
         9 . The expression vector of  claim 1  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 350 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 350 nucleotides.  
     
     
         10 . The expression vector of  claim 1  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 50 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 50 nucleotides.  
     
     
         11 . The expression vector of claims  8 ,  9  and  10  wherein said out-of-frame start codon is part of a consensus Kozak sequence.  
     
     
         12 . The expression vector of  claim 10  wherein said out-of-frame start codon and said translation initiation start site sequence are both included as part of a secondary structure.  
     
     
         13 . The expression vector of claims  8 ,  9 ,  10  wherein said translation initiation start site sequence is part of a secondary structure and said out-of-frame start codon is not part of said secondary structure.  
     
     
         14 . A dominant selectable marker encoded by a nucleic acid sequence, wherein the translation initiation start site of said dominant selectable marker is selected from the group consisting of TxxATGCxx; CxxATGCxx; CxxATGTxx; and TxxATGTxx, where “x” is a nucleotide, with the proviso that “Txx” downstream of the ATG codon does not encode a stop codon.  
     
     
         15 . The material of  claim 14  wherein said dominant selectable marker is selected from the group consisting of herpes simplex virus thymidine kinase, adenosine deaminase, asparagine synthetase, Salmonella his D gene, xanthine guanine phosphoribosyl transferase, hygromycin B phosphotransferase, and neomycin phosphotransferase.  
     
     
         16 . The material of  claim 14  wherein said translation initiation start site sequence is TxxATGCxx, where “x” is a nucleotide.  
     
     
         17 . The material of  claim 14  wherein said translation initiation start site sequence is TCCATGCTT.  
     
     
         18 . The material of  claim 14  wherein said translation initiation start site sequence is located within a secondary structure.  
     
     
         19 . The material of  claim 14  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 1000 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 1000 nucleotides.  
     
     
         20 . The material of  claim 14  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 350 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 350 nucleotides.  
     
     
         21 . The material of  claim 14  wherein said translation initiation start site sequence further comprises at least one out-of-frame start codon within about 50 nucleotides of the ATG start codon of said start site, with the proviso that no in-frame stop codon is located within said 50 nucleotides.  
     
     
         22 . The material of claims  19 ,  20  and  21  wherein said out-of-frame start codon is part of a consensus Kozak sequence.  
     
     
         23 . The material of  claim 21  wherein said out-of-frame start codon and said translation initiation start site sequence are both included as part of a secondary structure.  
     
     
         24 . The material of claims  19 ,  20 ,  21  wherein said translation initiation start site sequence is part of a secondary structure and said out-of-frame start codon is not part of said secondary structure.  
     
     
         25 . An expression vector selected from the group consisting of ANEX 1 (included within American Iype Culture Collection deposit number 69120) and ANEX 2 (included within ATCC deposit number 69118).  
     
     
         26 . A plasmid comprising the expression vector of  claim 1  wherein the nucleic acid sequence encoding for said protein of interest is co-linked to said dominant selectable marker.  
     
     
         27 . The plasmid of  claim 26  integrated within-the cellular deoxyribonucleic acid of a mammalian host cell.  
     
     
         28 . The plasmid of  claim 27  wherein said host cell is selected from the group consisting of DG44, DXB11, CV1, COS, R1610, SP2/0, P3×633-Ag8.653, BFA-1c1BPT, RAJI, and 293.  
     
     
         29 . The expression vector of  claim 1  further comprising an artificial intronic insertion region within said dominant selectable marker, wherein an encoding sequence for a protein of interest is located within said insertion region.  
     
     
         30 . The dominant selectable marker of  claim 14  further comprising an artificial instronic insertion region.