Expression constructs, host cells, and methods for producing insulin

Abstract

A cell for producing insulin includes an E. coli host cell and a polynucleotide designed for production of insulin disposed within the E. coli host cell. The polynucleotide includes a first inducible promoter and a first coding sequence to be transcribed from the first inducible promoter, the first coding sequence encoding an insulin polypeptide. The polynucleotide also includes a second inducible promoter and a second coding sequence to be transcribed from the second inducible promoter, the second coding sequence encoding one or more of cDsbA, cDsbC, a protein disulfide isomerase, Ervlp, and a chaperone. The first inducible promoter and the second inducible promoter are not responsive to the same inducer. A method of producing insulin includes growing a sample of the cells in a fermentation volume between 0.1 L and 1,000,000 L, adding an inducer of the first inducible promoter to induce expression of the insulin polypeptide, and purifying the insulin polypeptide.

What is claimed is: 
     
         1 . A cell for producing insulin, comprising:
 an  E. coli  host cell; and   a polynucleotide disposed within the  E. coli  host cell, the polynucleotide comprising:
 a first expression construct comprising a first inducible promoter and a first coding sequence to be transcribed from the first inducible promoter, the first coding sequence encoding an insulin polypeptide; and 
 a second expression construct comprising a second inducible promoter and a second coding sequence to be transcribed from the second inducible promoter, the second coding sequence encoding one or more of cDsbA, cDsbC, a protein disulfide isomerase, Ervlp, and a chaperone; and 
 wherein the first inducible promoter and the second inducible promoter are selected from the group consisting of  E. coli  sugar-inducible promoters and propionate-inducible promoters, and the first inducible promoter and the second inducible promoter are not responsive to the same inducer, and each of the first inducible promoter and the second inducible promoter is not a lactose-inducible promoter. 
   
     
     
         2 . The cell of  claim 1 , wherein the  E. coli  host cell has a reduced level of gene function of a first gene encoding a protein that metabolizes a first inducer of at least one of the first inducible promoter and the second inducible promoter. 
     
     
         3 . The cell of  claim 2 , wherein the gene is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB . 
     
     
         4 . The cell of  claim 1 , wherein the  E. coli  host cell has an altered level of gene function of a gene encoding a transporter protein for an inducer of at least one of the first inducible promoter and the second inducible promoter. 
     
     
         5 . The cell of  claim 1 , wherein the  E. coli  host cell is an  E. coli  EB0001 strain. 
     
     
         6 . The cell of  claim 1 , wherein at least one of the first inducible promoter and the second inducible promoter is selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, and a xylose-inducible promoter. 
     
     
         7 . The cell of  claim 1 , wherein each of the first inducible promoter and the second inducible promoter is independently selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, and a xylose-inducible promoter. 
     
     
         8 . The cell of  claim 1 , wherein the first inducible promoter is an L-arabinose-inducible promoter, and the second inducible promoter is a propionate-inducible promoter. 
     
     
         9 . The cell of  claim 1 , where the insulin polypeptide comprises at least one of an insulin A-chain and an insulin B-chain. 
     
     
         10 . The cell of  claim 1 , wherein the insulin polypeptide comprises both a mature A chain and a mature B chain. 
     
     
         11 . The cell of  claim 1 , wherein the insulin polypeptide comprises one of SEQ ID NO:4 and SEQ ID NO:9. 
     
     
         12 . The cell of  claim 1 , wherein the insulin polypeptide comprises one of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:12. 
     
     
         13 . The cell of  claim 1 , wherein the insulin polypeptide comprises SEQ ID NO:9 and SEQ ID NO:10. 
     
     
         14 . The cell of  claim 1 , wherein the second coding sequence encodes a chaperone. 
     
     
         15 . The cell of  claim 1 , wherein chaperone is selected from the group consisting of DnaK/DnaJ/GrpE, DsbC/DsbG, GroEL/GroES, IbpA/IbpB, Skp, Tig, and FkpA. 
     
     
         16 . The cell of  claim 1 , wherein the second coding sequence encodes Ervlp. 
     
     
         17 . A polynucleotide designed for production of insulin, the polynucleotide comprising:
 a first expression construct comprising a first inducible promoter and a first coding sequence to be transcribed from the first inducible promoter, the first coding sequence encoding an insulin polypeptide; and   a second expression construct comprising a second inducible promoter and a second coding sequence to be transcribed from the second inducible promoter, the second coding sequence encoding one or more of cDsbA, cDsbC, a protein disulfide isomerase, Ervlp, and a chaperone; and   wherein the first inducible promoter and the second inducible promoter are selected from the group consisting of  E. coli  sugar-inducible promoters and propionate-inducible promoters, and the first inducible promoter and the second inducible promoter are not responsive to the same inducer, and each of the first inducible promoter and the second inducible promoter is not a lactose-inducible promoter.   
     
     
         18 . A method of producing insulin, the method comprising;
 growing a sample of the cells of  claim 1  in a fermentation volume between 0.1 L and 1,000,000 L;   adding an inducer of the first inducible promoter, thereby inducing expression of the insulin polypeptide; and   purifying the insulin polypeptide.