Increased production of secreted proteins by recombinant eukaryotic cells

Abstract

Described herein are methods for increasing the amount of protein secreted by a cell. In one case, a cell is provided which contains a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted. In one case, the protein having unfolded protein response modulating activity is selected from the proteins selected from the group consisting of HAC1, PTC2 and lRE1. The protein of interest can be any secreted protein such as a therapeutic or an industrial enzyme. For example the protein can be selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.

What is claimed is:  
     
         1 . A method of increasing the secretion of a heterologous protein in a eukaryotic cell comprising inducing an elevated unfolded protein response (UPR).  
     
     
         2 . The method of    claim 1    wherein inducing is by increasing the presence of HAC1 protein in said cell.  
     
     
         3 . The method of    claim 2    wherein said HAC1 protein is constitutively produced.  
     
     
         4 . The method of    claim 2    wherein said increase of HAC1 protein is by a UPR inducing form of a HAC1 recombinant nucleic acid.  
     
     
         5 . The method of    claim 2    wherein said HAC1 protein is encoded by a nucleic acid isolated from a cell selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.  
     
     
         6 . The method of    claim 2    wherein said HAC1 protein is encoded by a nucleic acid isolated from yeast.  
     
     
         7 . The method of    claim 6    wherein said yeast is  Saccharomyces cerevisiae.    
     
     
         8 . The method of    claim 2    wherein said HAC1 protein is encoded by a nucleic acid isolated from filamentous fungi.  
     
     
         9 . The method of    claim 8    wherein said fungi is from Trichoderma.  
     
     
         10 . The method of    claim 8    wherein said fungi is  Trichoderma reesei.    
     
     
         11 . The method of    claim 8    wherein said fungi is from Aspergillus.  
     
     
         12 . The method of    claim 8    wherein said fungi is  Aspergillus nidulans.    
     
     
         13 . The method of    claim 8    wherein said fungi is  Aspergillus niger.    
     
     
         14 . The method of    claim 1    wherein said inducing is by modulating the level of lRE1 protein or PTC2 protein in said cell.  
     
     
         15 . The method of    claim 1    wherein said inducing is by increasing the level of lRE1 protein.  
     
     
         16 . The method of    claim 15    wherein said lRE1 is an lRE1 variant which has the activity of a constitutively phosphorylated lRE1.  
     
     
         17 . The method of    claim 15    wherein said lRE1 protein is encoded by a nucleic acid isolated from a cell selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.  
     
     
         18 . The method of    claim 15    wherein said lRE1 protein is encoded by a nucleic acid isolated from yeast.  
     
     
         19 . The method of    claim 18    wherein said yeast is  Saccharomyces cerevisiae.    
     
     
         20 . The method of    claim 15    wherein said lRE1 is isolated from filamentous fungi.  
     
     
         21 . The method of    claim 20    wherein said fungi is from Trichoderma.  
     
     
         22 . The method of    claim 20    wherein said fungi is  Trichoderma reesei.    
     
     
         23 . The method of    claim 20    wherein said fungi is from Aspergillus.  
     
     
         24 . The method of    claim 20    wherein said fungi is  Aspergillus nidulans.    
     
     
         25 . The method of    claim 20    wherein said fungi is  Aspergillus niger.    
     
     
         26 . The method of    claim 1    wherein said cell is selected from the group consisting of Aspergillus, Trichoderma, Saccharomyces, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Fusarium, Neurospora, and Penicillium.  
     
     
         27 . The method of    claim 1    wherein said cell is a yeast cell.  
     
     
         28 . The method of    claim 27    wherein said yeast is  Saccharomyces cerevisiae.    
     
     
         29 . The method of    claim 1    wherein said cell is from filamentous fungi.  
     
     
         30 . The method of    claim 29    wherein said fungi is from Trichoderma.  
     
     
         31 . The method of    claim 29    wherein said fungi is  Trichoderma reesei.    
     
     
         32 . The method of    claim 29    wherein said fungi is from Aspergillus.  
     
     
         33 . The method of    claim 29    wherein said fungi is  Aspergillus nidulans.    
     
     
         34 . The method of    claim 29    wherein said fungi is  Aspergillus niger.    
     
     
         35 . The method of    claim 1    wherein said cell is an insect cell.  
     
     
         36 . The method of    claim 1    wherein said cell is a mammalian cell.  
     
     
         37 . An isolated nucleic acid encoding a HAC1 protein, wherein said HAC1 protein induces unfolded protein response and has less than 50% similarity to yeast HAC1 protein.  
     
     
         38 . An isolated nucleic acid encoding a HAC1 protein, wherein said HAC1 protein induces unfolded protein response and wherein said HAC1 protein comprises a DNA binding region that has greater than 70% similarity to the DNA binding region of filamentous fungi HAC1 protein.  
     
     
         39 . The nucleic acid of    claim 38    wherein said filamentous fungi HAC1 protein has an amino acid sequence as shown in FIG. 7 or FIG. 8 or FIG. 28.  
     
     
         40 . The nucleic acid of    claim 38    wherein said HAC1 protein has an amino acid sequence having greater than 70% similarity to the sequence of FIG. 7 or FIG. 8 or FIG. 28.  
     
     
         41 . The nucleic acid of    claim 38    isolated from  Trichoderma reesei.    
     
     
         42 . The nucleic acid of    claim 38    isolated from  Aspergillus nidulans.    
     
     
         43 . The nucleic acid of    claim 38    isolated from  Aspergillus niger.    
     
     
         44 . The nucleic acid of    claim 38    wherein said HAC1 protein has an amino acid sequence as set forth in FIG. 7.  
     
     
         45 . The nucleic acid of    claim 38    wherein said HAC1 protein has an amino acid sequence as set forth in FIG. 8.  
     
     
         46 . The nucleic acid of    claim 38    wherein said HAC1 protein has an amino acid sequence as set forth in FIG. 28.  
     
     
         47 . The nucleic acid of    claim 38    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 7.  
     
     
         48 . The nucleic acid of    claim 38    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 7.  
     
     
         49 . The nucleic acid of    claim 38    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 8.  
     
     
         50 . The nucleic acid of    claim 38    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 8.  
     
     
         51 . The nucleic acid of    claim 38    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 28.  
     
     
         52 . The nucleic acid of    claim 38    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 28.  
     
     
         53 . A protein encoded by the nucleic acid of    claim 37   .  
     
     
         54 . A protein having unfolded protein response inducing activity and having greater than 70% similarity to an amino acid sequence of FIG. 7 or FIG. 8 or FIG. 28.  
     
     
         55 . A protein having an amino acid sequence as set forth in FIG. 7 or FIG. 8 or FIG. 28.  
     
     
         56 . An isolated nucleic acid encoding a PTC2 protein wherein said PTC2 protein modulates unfolded protein response and wherein said PTC2 has at least 70% similarity to an amino acid sequence of FIG. 24 or FIG. 25.  
     
     
         57 . The nucleic acid of    claim 56    isolated from  Trichoderma reesei.    
     
     
         58 . The nucleic acid of    claim 56    isolated from  Aspergillus nidulans.    
     
     
         59 . The nucleic acid of    claim 56    isolated from  Aspergillus niger.    
     
     
         60 . The nucleic acid of    claim 56    wherein said PTC2 protein has an amino acid sequence as set forth in FIG. 24.  
     
     
         61 . The nucleic acid of    claim 56    wherein said PTC2 protein has an amino acid sequence as set forth in FIG. 25.  
     
     
         62 . The nucleic acid of    claim 56    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 24.  
     
     
         63 . The nucleic acid of    claim 56    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 24.  
     
     
         64 . The nucleic acid of    claim 56    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 25.  
     
     
         65 . A protein encoded by the nucleic acid of    claim 56   .  
     
     
         66 . A protein having unfolded protein response modulating activity and having greater than 70% similarity to the amino acid sequence of FIG. 24 or FIG. 25.  
     
     
         67 . A protein having an amino acid sequence as set forth in FIG. 24 or FIG. 25.  
     
     
         68 . A nucleic acid encoding a lRE1 protein having unfolded protein response modulating activity and having at least 60% to an amino acid sequence as shown in FIG. 26 or FIG. 27.  
     
     
         69 . The nucleic acid of    claim 68    wherein said lRE1 protein has an amino acid sequence as shown in FIG. 26 or FIG. 27.  
     
     
         70 . The nucleic acid of    claim 68    wherein said nucleic acid is isolated from  Trichoderma reesei.    
     
     
         71 . The nucleic acid of    claim 68    wherein said nucleic acid is isolated from  Aspergillus nidulans.    
     
     
         72 . The nucleic acid of    claim 68    wherein said nucleic acid is isolated from  Aspergillus niger.    
     
     
         73 . The nucleic acid of    claim 68    wherein said lRE1 protein has an amino acid sequence as set forth in FIG. 26.  
     
     
         74 . The nucleic acid of    claim 68    wherein said lRE1 protein has an amino acid sequence as set forth in FIG. 27.  
     
     
         75 . The nucleic acid of    claim 68    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 26.  
     
     
         76 . The nucleic acid of    claim 68    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 26.  
     
     
         77 . The nucleic acid of    claim 68    wherein said nucleic acid comprises a coding nucleic acid sequence as set forth in FIG. 27.  
     
     
         78 . The nucleic acid of    claim 68    wherein said nucleic acid consists essentially of a coding nucleic acid sequence as set forth in FIG. 27.  
     
     
         79 . A protein encoded by the nucleic acid of    claim 68   .  
     
     
         80 . A protein having unfolded protein response inducing activity and having greater than 70% similarity to the amino acid sequence of FIG. 26 or FIG. 27.  
     
     
         81 . The protein of    claim 80    wherein said protein has constitutive unfolded protein response inducing activity.  
     
     
         82 . A protein having an amino acid sequence as set forth in FIG. 26 or FIG. 27.  
     
     
         83 . A cell containing a heterologous nucleic acid encoding a protein having unfolded protein response modulating activity and a heterologous nucleic acid encoding a protein of interest to be secreted.  
     
     
         84 . The cell of    claim 83    wherein said protein having unfolded protein response modulating activity is selected from the proteins selected from the group consisting of HAC1, PTC2 and lRE1.  
     
     
         85 . The cell of    claim 83    wherein said protein of interest is selected from the group consisting of lipase, cellulase, endo-glucosidase H, protease, carbohydrase, reductase, oxidase, isomerase, transferase, kinase, phosphatase, alpha-amylase, glucoamylase, lignocellulose hemicellulase, pectinase and ligninase.  
     
     
         86 . A protein encoded by the nucleic acid of    claim 38   .