Methods for improving recombinant protein expression

Abstract

Materials and methods are provided which allowed for increased expression of a transfected gene of interest in a recombinant host cell.

1 . A method for increasing heterologous protein expression in a host cell comprising the steps of culturing the host cell comprising a first heterologous polynucleotide sequence encoding said heterologous protein under conditions that allow for protein expression, said first polynucleotide encoded on a vector, said host cell further comprising a second polynucleotide sequence having a protein coding sequence for a selectable marker protein, said second polynucleotide having a sequence modification compared to a wild-type polynucleotide encoding said selectable marker protein, said sequence modification reducing translation efficiency of mRNA encoded by said second polynucleotide, said sequence modification, said second polynucleotide having said sequence modification and said wild-type polynucleotide encoding identical amino acid sequences for said selectable marker protein. 
     
     
         2 . The method of  claim 1  wherein said first polynucleotide and said second polynucleotide are in a single vector. 
     
     
         3 . The method of  claim 2  wherein the first polynucleotide and second polynucleotide are each under transcriptional control of distinct promoters. 
     
     
         4 . The method of  claim 2  wherein the first polynucleotide and second polynucleotide are each under transcriptional control of a single promoter. 
     
     
         5 . The method of  claim 1  wherein said first polynucleotide and said second polynucleotide are in separate vectors. 
     
     
         6 . The method of  claim 1  wherein the modification is in an untranslated region of said second polynucleotide encoding said selectable marker protein. 
     
     
         7 .- 8 . (canceled) 
     
     
         9 . The method of  claim 1  wherein the modification is in a protein coding region of the gene encoding the selectable marker protein. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1  wherein said protein coding sequence in said second polynucleotide sequence comprises at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding said selectable marker protein, said modified codon being a codon that is not a preferred codon for the encoded amino acid for the host cell. 
     
     
         12 . (canceled) 
     
     
         13 . The method of  claim 1  wherein said protein coding sequence in said second polynucleotide sequence comprising at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding said selectable marker protein, and said modification introduces a change in secondary structure of said mRNA which reduces translation efficiency of said mRNA. 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 1  wherein said protein coding sequence in said second polynucleotide sequence comprising at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding said selectable marker protein, and said modification modifies G+C content of said mRNA 
     
     
         16 .- 17 . (canceled) 
     
     
         18 . The method of  claim 1  wherein said protein coding sequence in said second polynucleotide sequence comprising at least one modified codon that is not a wild-type codon in a wild-type polynucleotide encoding said selectable marker protein, and said modification modifies A+T content of said mRNA 
     
     
         19 .- 21 . (canceled) 
     
     
         22 . The method of  claim 1 , wherein said selectable marker protein is selected from the group consisting of neomycin phosphotransferase (npt II), hygromycin phosphotransferase (hpt), dihydrofoate reductase (dhfr), zeocin, phleomycin, bleomycin resistance gene ble (enzyme not known), gentamycin acetyltransferase, streptomycin phosphotransferase, mutant form of acetolactate synthase (als), bromoxynil nitrilase, phosphinothricin acetyl transferase (bar), enolpyruvylshikimate-3-phosphate (EPSP) synthase (aro A), muscle specific tyrosine kinase receptor molecule (MuSK-R), copper-zinc superoxide dismutase (sod1), metallothioneins (cup1, MT1), beta-lactamase (BLA), puromycin N-acetyl-transferase (pac), blasticidin acetyl transferase (bls), blasticidin deaminase (bsr), histidinol dehydrogenase (HDH), N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthetase (ade1), argininosuccinate lyase (arg4), beta-isopropylmalate dehydrogenase (leu2), invertase (suc2) and orotidine-5′-phosphate (OMP) decarboxylase (ura3). 
     
     
         23 . The method of  claim 1 , wherein the host cell is selected from the group consisting of: (a) a eukaryotic cell; (b) a prokaryotic cell; (c) a yeast cell; (d) an insect cell; (e) a plant cell; (f) a protozoan cell; and (g) a mammalian cell. 
     
     
         24 . (canceled) 
     
     
         25 . The method of  claim 23  wherein the host cell is  Escherichia  coli. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 23 , wherein the host cell is  Saccharomyces cerevisiae.    
     
     
         28 . The method of  claim 23 , wherein the host cell is  Pichia pastoris.    
     
     
         29 . (canceled) 
     
     
         30 . The method of  claim 23 , wherein the host cell is  Spodoptera frugiperda.    
     
     
         31 .- 33 . (canceled) 
     
     
         34 . The method of  claim 23  wherein the host cell is a human cell. 
     
     
         35 . The method of  claim 23  wherein said host cell is of Chinese hamster cell. 
     
     
         36 . The method of  claim 35  wherein said host cell is a Chinese hamster ovary cell. 
     
     
         37 . The method of  claim 1 , wherein the expression vector is a Chinese hamster elongation factor 1 (CHEF1) expression vector. 
     
     
         38 . The method of  claim 1  wherein the second polynucleotide comprises the polynucleotide set out in  FIG. 2 . 
     
     
         39 . The method of  claim 38  wherein the expression vector is a Chinese hamster elongation factor 1 (CHEF1) expression vector.