Methods and Compositions for Increasing Protein Production

Abstract

The disclosure provides methods and materials for increasing the expression of a protein of interest such as an antibody by a cell. ABC50 expression or activity is increased which increases expression of the protein or antibody of interest. The disclosure also provides methods and materials for increasing the sensitivity of a cell to an endoplasmic reticulum stress agent such as Econozole by decreasing the level of ABC50.

1 .- 28 . (canceled) 
     
     
         29 . A process for the production of a protein of interest comprising: a) increasing production of a protein of interest in a eukaryotic cell according to  claim 46 ; b) culturing the eukaryotic cell under suitable conditions permitting the expression of the protein of interest and the ABC50 protein or fragment; culturing the cell until the protein of interest accumulates and isolating the protein of interest. 
     
     
         30 . The process of  claim 29  wherein the protein of interest is an antibody or fragment thereof. 
     
     
         31 .- 33 . (canceled) 
     
     
         34 . A vector comprising a polynucleotide encoding an ABC50 polynucleotide and optionally a polynucleotide encoding a protein of interest, wherein the polynucleotide(s) is/are operably linked to one or more promoters. 
     
     
         35 . The vector of  claim 34 , wherein the vector is a retroviral vector, optionally a lentiviral vector. 
     
     
         36 . A eukaryotic cell a) comprising the vector of  claim 34  expressing increased ABC50 protein and/or activity compared to a control cell not comprising said vector; or b) resistant to Econozole (Ec), wherein the Ec resistant cell has increased ABC50 protein levels and/or activity compared to a non-Ec resistant control cell, wherein the eukaryotic cell is suitable and/or adapted for expression of a protein of interest. 
     
     
         37 . (canceled) 
     
     
         38 . The cell of  claim 36 , wherein the eukaryotic cell is selected from a yeast, plant, worm, insect, avian, fish, reptile, mammalian, hybridoma, a myeloma cell or a spleen cell. 
     
     
         39 . A system for heterologous protein expression comprising the eukaryotic cell of  claim 36  b) and an expression vector comprising a multicloning site for receiving a heterologous polynucleotide encoding the heterologous protein to be expressed. 
     
     
         40 .- 42 . (canceled) 
     
     
         43 . The method of claim  42 , wherein the shRNA comprises SEQ ID NO: 3 and/or 4. 
     
     
         44 . The method of claim  41  wherein the ER stress agent is selected from EC, thapsigargin and tunicamycin. 
     
     
         45 . A composition comprising: a polynucleotide comprising SEQ ID NO:3 and/or 4, an isolated vector comprising a polynucleotide encoding an ABC50 polynucleotide and optionally a polynucleotide encoding a protein of interest, wherein the polynucleotide(s) is/are operably linked to one or more promoters, a eukaryotic cell comprising said vector or a eukaryotic cell that is Ec resistant and has increased protein levels and/or activity compared to a non-Ec resistant control cell, and/or an isolated protein produced according to the method of  claim 46 . 
     
     
         46 . A method for increasing the production of a protein of interest, said method comprising:
 I)
 a) introducing into a eukaryotic cell a polynucleotide encoding an ABC50 protein or active fragment thereof having protein synthesis increasing activity and/or eIF2 binding activity and optionally encoding a selectable marker into a eukaryotic cell, 
 wherein said eukaryotic cell expresses a protein of interest or wherein after the introduction of the polynucleotide encoding an ABC50 protein or active fragment the eukaryotic cell is fused to a second eukaryotic cell expressing a protein of interest, thus producing a eukaryotic cell expressing ABC50 protein or active fragment thereof and the protein of interest, and 
 b) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest in the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein; 
   II)
 a), chemically inducing the expression of endogenous ABC50 protein in a eukaryotic cell, 
 wherein said eukaryotic cell expresses a protein of interest or wherein after the chemical induction of the polynucleotide encoding an ABC50 protein the eukaryotic cell is fused to a second eukaryotic cell expressing a protein of interest, thus producing a eukaryotic cell expressing ABC50 protein and a protein of interest, and 
 b) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest in the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein; 
   III)
 a) introducing into a eukaryotic cell a polynucleotide encoding a protein of interest and optionally encoding a selectable marker, 
 wherein said eukaryotic cell expresses an ABC50 protein or active fragment thereof having protein synthesis increasing activity and/or eIF2 binding activity, 
 wherein said ABC50 protein or active fragment thereof is recombinantly expressed in the eurkaryotic cell after the introduction of the polynucleotide encoding an ABC50 protein or active fragment thereof, thus producing a eukaryotic cell expressing ABC50 protein or active fragment thereof and a protein of interest, and 
 b) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest in the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein; 
   IV)
 a) introducing into a eukaryotic cell a polynucleotide encoding a protein of interest and optionally encoding a selectable marker, 
 wherein said eukaryotic cell expresses an ABC50 protein, 
 wherein said ABC50 protein is expressed by chemically inducing the expression of endogenous ABC50 protein in the eukaryotic cell, thus producing a eukaryotic cell expressing ABC50 protein or active fragment thereof and a protein of interest, and 
 b) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest in the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein; 
   V)
 a) introducing into a eukaryotic cell a polynucleotide encoding an ABC50 protein or active fragment thereof having protein synthesis increasing activity and/or eIF2 binding activity and optionally encoding a selectable marker into a eukaryotic cell, 
 b) introducing into the eukaryotic cell produced in step a) a polynucleotide encoding a protein of interest and optionally encoding a selectable marker, 
 thus producing a eukaryotic cell expressing ABC50 protein or active fragment thereof and a protein of interest, and 
 c) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest in the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein; or 
   VI)
 a) chemically inducing the expression of endogenous ABC50 protein in a eukaryotic cell, and 
 b) introducing into the eukaryotic cell produced in step a) a polynucleotide encoding a protein of interest and optionally encoding a selectable marker, 
 thus producing a eukaryotic cell expressing ABC50 protein or active fragment thereof and a protein of interest, and 
 c) incubating the eukaryotic cell under suitable conditions for a suitable length of time to produce the protein of interest and the presence of the ABC50 protein such that the protein of interest is produced at higher levels than in the absence of the ABC50 protein. 
   
     
     
         47 . The method of  claim 46 , wherein the protein of interest is a heterologous protein. 
     
     
         48 . The method of  claim 46 , wherein the method further comprises isolating the protein of interest, optionally wherein protein of interest is secreted and is secreted into a culture medium, the method further comprising isolating the secreted protein from the culture medium, or wherein the protein of interest is intracellular, the method further comprising lysing the cell and isolating the intracellular protein of interest, or wherein the protein of interest is membrane or surface bound, the method further comprising solubilizing the cell membrane and isolating the membrane protein or surface bound protein of interest. 
     
     
         49 . The method of  claim 46 , wherein the ABC50 protein comprises SEQ ID NO: 1, 2 or 5; or a protein with at least 85%, 88%, 90%, 95%, 99% or 99.5% sequence identity with SEQ ID NO:1, 2 or 5. 
     
     
         50 . The method of  claim 46 , wherein the increased expression is about 5% to about 10%, about 11% to about 20%, about 21% to about 30%, about 31% to about 40%, about 41% to about 50%, 51% to about 60%, 61% to about 70%, 71% to about 80%, about 81% to about 90%, about 91% to about 100%, about 150% to about 199%, about 200% to about 299%, about 300% to about 499%, or about 500% to about 1000%. 
     
     
         51 . The method of  claim 46 , wherein the eukaryotic cell is selected from a yeast, plant, worm, insect, avian, fish, reptile and mammalian cell. 
     
     
         52 . The method of  claim 46 , wherein the selectable marker is selected from β-galactosidase, chloramphenicol acetyltransferase, firefly luciferase, an antibiotic resistance gene neomycin and hygromycin, dihydrofolate reductase (DHFR) and glutamine synthetase (GS) and optionally wherein selection comprises amplification of the integrated DNA by exposure of the selected cells to methotrexate (MTX) or methionine sulphoximine (MSX). 
     
     
         53 . The method of  claim 52 , wherein the antibiotic resistance gene is selected from neomycin and hygromycin. 
     
     
         54 . The method of  claim 46 , wherein the ABC50 protein is chemically increased by induction of econazole resistance and selecting for ABC50 expressing cells.