Methods and compositions for expression of polypeptides in a cell

Abstract

Disclosed herein are vector systems for expression of polypeptides in eukaryotic cells; and methods of obtaining high-level expression of polypeptides in a eukaryotic cell. Methods and compositions for obtaining stable, long-term expression of recombinant polypeptides are also provided

What is claimed is: 
     
         1 . A polynucleotide comprising:
 (a) a cytomegalovirus (CMV) RC-UTR downstream sequence,   (b) a CMV major immediate early (MIE) promoter,   (c) a CMV UTR upstream sequence,   (d) a CMV Intron A, and   (e) a CMV UTR downstream sequence.   
     
     
         2 . The polynucleotide of  claim 1 , wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO:1 or a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1. 
     
     
         3 . A nucleic acid vector comprising:
 (a) the polynucleotide of  claim 1 ; and   (b) a polyadenylation signal.   
     
     
         4 . The vector of  claim 3 , wherein the polyadenylation signal is selected from the group consisting of the simian virus 40 (SV40) polyadenylation signal and the bovine growth hormone (BGH) polyadenylation signal. 
     
     
         5 . The vector of  claim 4 , further comprising a multiple cloning site disposed between the CMV sequences and the polyadenylation signal. 
     
     
         6 . The vector of  claim 5 , further comprising a transgene. 
     
     
         7 . The vector of  claim 5 , further comprising a post-transcriptional regulatory element (PRE). 
     
     
         8 . The vector of  claim 7 , wherein the PRE is selected from the group consisting of a woodchuck hepatitis virus PRE (WPRE), a human hepatitis B virus PRE (HPRE), and hybrids thereof. 
     
     
         9 . The vector of  claim 6 , further comprising a MAR/SAR sequence. 
     
     
         10 . The vector of  claim 9 , wherein the MAR/SAR sequence is selected from the group consisting of a chicken lysozyme MAR/SAR (CLM) sequence, an interferon alpha-2 MAR/SAR (IAM) sequence, an interferon beta MAR/SAR (IBM) sequence, a X29 MAR/SAR sequence, a S4 MAR/SAR sequence and hybrids thereof. 
     
     
         11 . The vector of  claim 6 , wherein the transgene is a gene that encodes a protein selected from the group consisting a recombinant protein, a fusion protein, an antibody, a cytokine, a hormone, an enzyme and a clotting factor. 
     
     
         12 . A cell comprising the vector of  claim 6 , wherein the cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell. 
     
     
         13 . A method for expressing a polypeptide in a population of transfected cells, the method comprising:
 transfecting a population of cells with the vector of  claim 13  under conditions such that greater than 20% of the cells in the population express a polypeptide encoded by a transgene.   
     
     
         14 . The method of  claim 13 , wherein the cells expressing the polypeptide express the polypeptide for at least twenty-five generations. 
     
     
         15 . The method of  claim 13 , wherein the cells expressing the polypeptide express the polypeptide for at least fifty generations.