Vectors for use in an inducible coexpression system

Abstract

The present invention provides expression vectors for use in an inducible coexpression system, capable of controlled induction of expression of each gene product.

What is claimed is: 
     
         1 . An expression construct comprising two or more inducible promoters, wherein at least one of said inducible promoters is responsive to an inducer that is different than the inducer of another of said inducible promoters, and wherein none of the inducible promoters is an inducible promoter selected from the group consisting of: a tetracycline-inducible promoter, a copper-inducible promoter, and a methionine-inducible promoter. 
     
     
         2 . The expression construct of  claim 1 , wherein the expression construct is extrachromosomal. 
     
     
         3 . The expression construct of  claim 1 , wherein each inducible promoter is selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, and a promoter inducible by phosphate depletion. 
     
     
         4 . The expression construct of  claim 3 , wherein at least one inducible promoter is selected from the group consisting of the araBAD promoter, the prpBCDE promoter, the rhaSR promoter, the xlyA promoter, the lacZYA promoter, and the phoA promoter. 
     
     
         5 . The expression construct of  claim 1 , wherein the expression construct comprises at least one propionate-inducible promoter and at least one inducible promoter selected from the group consisting of an L-arabinose-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, and a promoter inducible by phosphate depletion. 
     
     
         6 . The expression construct of  claim 5 , wherein the propionate-inducible promoter is the prpBCDE promoter. 
     
     
         7 . The expression construct of  claim 5  comprising a nucleotide sequence having at least 80% sequence identity to at least 50 contiguous bases of nucleotides 4937 through 5185 of SEQ ID NO:15. 
     
     
         8 . The expression construct of  claim 1 , wherein at least one inducible promoter is a propionate-inducible promoter and at least one other inducible promoter is an L-arabinose-inducible promoter. 
     
     
         9 . The expression construct of  claim 8  comprising a nucleotide sequence having at least 80% sequence identity to at least 50 contiguous bases of nucleotides 2818 through 3151 of SEQ ID NO:15, and at least 80% sequence identity to at least 50 contiguous bases of nucleotides 4937 through 5185 of SEQ ID NO:15. 
     
     
         10 . The expression construct of  claim 9  comprising SEQ ID NO:15. 
     
     
         11 . A host cell comprising the expression construct of  claim 1 . 
     
     
         12 . The host cell of  claim 11  wherein the host cell is a prokaryotic cell. 
     
     
         13 . The host cell of  claim 12  wherein the host cell is  E. coli.    
     
     
         14 . The host cell of  claim 13  wherein the host cell further comprises one or more of the following: (a) a deletion of the araBAD genes; (b) an altered gene function of the araE and araFGH genes; (c) a lacY(A177C) gene; (d) a reduced gene function of the prpB and prpD genes; (e) a reduced gene function of the sbm/scpA-ygfD/argK-ygfGH/scpBC genes; (f) a reduced gene function of the gor and trxB genes; (g) a reduced gene function of the AscG gene; (h) a polynucleotide encoding a form of DsbC lacking a signal peptide; (i) a polynucleotide encoding Erv1p; and (j) a polynucleotide encoding a chaperone. 
     
     
         15 . The host cell of  claim 13  wherein the host cell is  E. coli  ASE(DGH). 
     
     
         16 . A host cell comprising the expression construct of  claim 1 , wherein the expression construct further comprises at least one polynucleotide sequence encoding at least one gene product to be transcribed from an inducible promoter. 
     
     
         17 . A method of producing a gene product, the method comprising growing a culture of the host cell of  claim 16  and adding an inducer of at least one inducible promoter to the culture. 
     
     
         18 . The method of  claim 17 , wherein the method further comprises purifying the gene product from the culture. 
     
     
         19 . A gene product produced by the method of  claim 17 . 
     
     
         20 . The gene product of  claim 19  wherein the gene product is selected from the group consisting of: (a) an immunoglobulin heavy chain; (b) an immunoglobulin light chain; and (c) a fragment of any of (a)-(b). 
     
     
         21 . The gene product of  claim 20 , wherein the gene product is part of a multimeric product. 
     
     
         22 . The multimeric product of  claim 21 , wherein the multimeric product is an antibody. 
     
     
         23 . A kit comprising the host cell of  claim 11 . 
     
     
         24 . An expression construct comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence having at least 97% sequence identity to at least 225 contiguous bases of nucleotides 4937 through 5185 of SEQ ID NO:15;   (b) a nucleotide sequence having at least 80% sequence identity to at least 300 contiguous bases of nucleotides 4937 through 5304 of SEQ ID NO:15;   (c) a nucleotide sequence having at least 87% sequence identity to at least 350 contiguous bases of nucleotides 2818 through 3259 of SEQ ID NO:15; and   (d) a nucleotide sequence having at least 90% sequence identity to at least 450 contiguous bases of nucleotides 10 through 1822 of SEQ ID NO:2.