Compositions and Methods Related to mRNA Translational Enhancer Elements

Abstract

Provided are mRNA translational enhancer elements (TEEs), e.g., SEQ ID NOs:1-35. Also provided are translational enhancer polynucleotides that comprise one or more of the specific TEEs exemplified herein or their variants, homologs or functional derivatives. Further provided are expression vectors comprising such TEEs or translational enhancer polynucleotides, as well as host cells and expression systems that harbor such vectors.

What is claimed is: 
     
         1 . A cloning or expression vector comprising the following elements: (1) a transcriptional promoter, (2) a polynucleotide sequence having a eukaryotic translation initiation codon and encoding a polypeptide operably linked to the transcriptional promoter, (3) a 5′ untranslated sequence located between the transcriptional promoter and the eukaryotic translation initiation codon, and (4) a translational enhancer polynucleotide located within the 5′ untranslated sequence,
 wherein the translational enhancer polynucleotide comprises at least two translational enhancer elements (TEEs) and one or more optional spacer sequences of 1 to 100 nucleotides in length, said at least two TEEs consisting of the sequence R 5 M 3 S 2 CS 3 GCN 2 GM 4 WR 6  (SEQ ID NO: 4) wherein: 
 R 5  is absent, or A if present; 
 M 3  is absent, or C or A if present; 
 S 2  is G; 
 S 3  is G or C, and S 2  and S 3  can be the same or different; 
 N 2  is G, C or T; 
 M 4  is A or C; 
 W is absent, or A or T if present; and 
 R 6  is absent, or A if present,
 wherein the said at least two TEEs are located within about 1-500 nucleotides 5′ of the eukaryotic translation initiation codon, and 
 wherein the cloning or expression vector exhibits increased cap-dependent translation compared to the same vector lacking the translational enhancer polynucleotide. 
 
 
     
     
         2 . The cloning or expression vector of  claim 1 , wherein said at least two TEEs consists of at least two sequences selected from the group consisting of one or more of the following sequences: 5′-CGCGGCTGA-3′ (SEQ ID NO: 31); 5′-AGCCGCCGCA-3′ (SEQ ID NO:34); and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         3 . The cloning or expression vector of  claim 1 , wherein said at least two TEEs consists of at least two sequences that are at least 90% identical to a sequence selected from the group consisting of the following sequences: 5′-CGCGGCTGA-3′ (SEQ ID NO: 31); 5′-AGCCGCCGCA-3′ (SEQ ID NO:34); and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         4 . The cloning or expression vector of  claim 1 , wherein said at least two TEEs consists of at least two sequences that are at least 95% identical to one or more sequences selected from the group consisting of 5′-CGCGGCTGA-3′ (SEQ ID NO: 31), 5′-AGCCGCCGCA-3′ (SEQ ID NO:34), and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         5 . The cloning or expression vector of  claim 1 , wherein said translational enhancer polynucleotide comprises at least five copies of the same translational enhancer element (TEE). 
     
     
         6 . The cloning or expression vector of  claim 1 , wherein said translational enhancer polynucleotide comprises at least ten copies of the same translational enhancer element (TEE). 
     
     
         7 . The cloning or expression vector of  claim 1 , wherein the said at least two TEEs are identical to each other. 
     
     
         8 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is at least 90% identical to a sequence selected from the group consisting of 5′-CGCGGCTGA-3′ (SEQ ID NO: 31), 5′-AGCCGCCGCA-3′ (SEQ ID NO:34), and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         9 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is at least 95% identical to a sequence selected from the group consisting of 5% CGCGGCTGA-3′ (SEQ ID NO: 31), 5′-AGCCGCCGCA-3′ (SEQ ID NO:34), and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         10 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is identical to a sequence selected from the group consisting of 5% CGCGGCTGA-3′ (SEQ ID NO: 31), 5′-AGCCGCCGCA-3′ (SEQ ID NO:34), and 5′-ACGCCGCCGA-3′ (SEQ ID NO:35). 
     
     
         11 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is identical to 5′-CGCGGCTGA-3′ (SEQ ID NO: 31). 
     
     
         12 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is identical to 5′-CGCGGCTGA-3′ (SEQ ID NO: 34). 
     
     
         13 . The cloning or expression vector of  claim 5 , wherein said TEE consists of a sequence that is identical to 5′-CGCGGCTGA-3′ (SEQ ID NO: 35). 
     
     
         14 . A DNA vaccine comprising the cloning or expression vector of  claim 1 . 
     
     
         15 . A eukaryotic host cell transfected with the cloning or expression vector of  claim 1 . 
     
     
         16 . The eukaryotic host cell of  claim 15 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell. 
     
     
         17 . A method for recombinantly producing a polypeptide comprising:
 (i) constructing the cloning or expression vector of  claim 1 ;   (ii) transfecting said vector into a eukaryotic host cell; and   (iii) culturing the host cell transfected with said vector; thereby producing the polypeptide.   
     
     
         18 . The method of  claim 17 , further comprising a step of purifying the recombinant polypeptide.