High level expression of recombinant toxin proteins

Abstract

The present invention relates to the field of recombinant toxin protein production in bacterial hosts. In particular, the present invention relates to production processes for obtaining high levels of a recombinant CRM197, Diphtheria Toxin, Pertussis Toxin, Tetanus Toxoid Fragment C, Cholera Toxin B, Cholera holotoxin, and Pseudomonas Exotoxin A, from a bacterial host.

What is claimed is: 
     
         1 . A method for producing a recombinant toxin protein in a Pseudomonad host cell, said method comprising:
 ligating into an expression vector a nucleotide sequence encoding the toxin protein;   transforming the Pseudomonad host cell with the expression vector; and   culturing the transformed Pseudomonad host cell in a culture media suitable for the expression of the recombinant toxin protein;   wherein the recombinant toxin protein is CRM197, Diphtheria Toxin, Cholera holotoxin, Cholera Toxin B, Pertussis Toxin, Tetanus Toxin Fragment C,  C. difficile  Toxin B, or  P. aeruginosa  Exotoxin A.   
     
     
         2 . The method of  claim 1 , wherein the recombinant protein is produced at a yield of soluble and/or active toxin protein of 0.2 grams per liter to about 12 grams per liter. 
     
     
         3 . The method of  claim 1 , wherein the nucleotide sequence encoding the toxin protein is fused to a secretion signal coding sequence that when expressed directs transfer of the toxin protein to the periplasm. 
     
     
         4 . The method of  claim 1 , wherein the host cell is defective in the expression of at least one protease or wherein the host cell overexpresses at least one folding modulator, or a combination thereof. 
     
     
         5 . The method of  claim 1 , wherein the recombinant toxin protein is CRM197 and the host cell is defective in the expression of Hs1U, Hs1V, Prc1, DegP1, DegP2, and AprA. 
     
     
         6 . The method of  claim 5 , wherein the recombinant toxin protein is fused to a secretion leader that is Azu, IbpS31A, CupA2, PbpA20V, or Pbp. 
     
     
         7 . The method of  claim 1 , wherein the recombinant toxin protein is CRM197 and the host cell is defective in the expression of Serralysin, Hs1U, Hs1V, Prc1, DegP1, DegP2, or AprA, or a combination thereof, or wherein the host cell overexpresses DsbA, DsbB, DsbC, and DsbD, and further wherein the recombinant toxin protein is fused to the Azu, Pbp, or native secretion leader. 
     
     
         8 . The method of  claim 1 , wherein the recombinant toxin protein is CRM197, the host cell is wild-type and wherein the recombinant toxin protein is fused to the secretion leader Pbp or Azu. 
     
     
         9 . The method of  claim 1 , wherein the recombinant toxin protein is CRM197 and wherein the recombinant toxin protein is fused to the secretion leader Azu, Pbp, IbpS31A, CupA2, or PbpA20V. 
     
     
         10 . The method of  claim 1 , wherein the recombinant toxin protein is Cholera Toxin B and the host cell: is defective in the expression of Lon, La, and AprA, or; is defective in the expression of Hs1U, Hs1V, Prc1, DegP1, DegP2, and AprA, and overexpresses DegP2 S219A. 
     
     
         11 . The method of  claim 10 , wherein the host cell: is defective in the expression of Lon, La, and AprA and wherein the recombinant toxin protein is fused to the secretion leader Pbp A20V, or; is defective in the expression of Hs1U, Hs1V, Prc1, DegP1, DegP2, and AprA, overexpresses DegP2 S219A, and wherein the recombinant toxin protein is fused to the secretion leader DsbA. 
     
     
         12 . The method of  claim 1 , wherein the recombinant toxin protein is Pertussis toxin S1 E129A R9K and the host cell: is defective in the expression of: Lon, La, and AprA; overexpresses GrpE, DnaK, and DnaJ; is defective in the expression of HtpX; is defective in the expression of RXF01590; or is defective in the expression of ppiB (RXF05345). 
     
     
         13 . The method of  claim 12 , wherein the recombinant toxin protein is fused to its native secretion leader. 
     
     
         14 . The method of  claim 1 , wherein the recombinant toxin protein is Tetanus Toxin C and the host cell is defective in the expression of Lon, La and AprA, or the host cell is defective in the expression of Hs1U, Hs1V, Prc1, DegP1, DegP2, and AprA, or the host cell overexpresses dsbABCD, or the host cell overexpresses GrpE, DnaK, and DnaJ. 
     
     
         15 . The method of  claim 14 , wherein the recombinant toxin protein is fused to the secretion leader TolB, DsbA, DsbC, Pbp A20V, NikA, or CupA2. 
     
     
         16 . The method of  claim 1 , wherein the recombinant toxin protein is  C. difficile  Toxin B and the host cell: is defective in the expression of HtpX; is defective in the expression of DegP1; is defective in the expression of Hs1U, Hs1V, Prc1 and Prc2; is defective in the expression of Lon, la, and DegP2, or; the host cell is defective in the expression of Lon, Prc1, DegP2, AprA and overexpresses DegP2 S219A. 
     
     
         17 . The method of  claim 1 , further comprising measuring the activity of the recombinant toxin protein in an activity assay, wherein about 40% to about 100% of the soluble toxin protein produced is determined to be active. 
     
     
         18 . The method of  claim 17 , wherein the activity assay is an immunological assay, a receptor-binding assay, or an enzyme assay. 
     
     
         19 . The method of  claim 1 , wherein the expression vector comprises a lac derivative promoter operatively linked to the protein coding sequence, and wherein the culturing comprises induction of the promoter using IPTG at a concentration of about 0.02 to about 1.0 mM, the cell density at induction is an optical density of about 40 to about 200 absorbance units (AU), the pH of the culture is from about 6 to about 7.5, and the growth temperature is about 20 to about 35° C. 
     
     
         20 . The method of  claim 1 , wherein the host cell is a  Pseudomonas  cell. 
     
     
         21 . The method of  claim 1 , wherein the host cell is  Pseudomonas fluorescens.    
     
     
         22 . The method of  claim 1 , wherein the nucleotide sequence has been optimized for expression in the Pseudomonad host cell. 
     
     
         23 . The method of  claim 20 , wherein the nucleotide sequence has been optimized for expression in the  Pseudomonas  host cell. 
     
     
         24 . The method of  claim 23 , wherein the nucleotide sequence has been optimized for expression in the  Pseudomonas fluorescens  host cell. 
     
     
         25 . The method of  claim 1 , wherein the Pertussis Toxin is wild-type or S1 E129A R9K. 
     
     
         26 . The method of  claim 1 , wherein the  P. aeruginosa  Exotoxin A is wild-type, CRM66, or rEPA. 
     
     
         27 . The method of  claim 3 , wherein the expression vector further comprises a tag sequence adjacent to the coding sequence for the secretion signal. 
     
     
         28 . The method of  claim 1 , wherein the expression vector further comprises a tag sequence adjacent to the coding sequence for the toxin protein. 
     
     
         29 . A recombinant toxin protein produced in a Pseudomonad host cell, wherein said recombinant toxin protein is produced by a method comprising:
 ligating into an expression vector a nucleotide sequence encoding the toxin protein;   transforming the Pseudomonad host cell with the expression vector; and   culturing the transformed Pseudomonad host cell in a culture media suitable for the expression of the recombinant toxin protein;   wherein the recombinant toxin protein is CRM197, Diphtheria Toxin, Cholera holotoxin, Cholera Toxin B, Pertussis Toxin, Tetanus Toxin Fragment C,  C. difficile  Toxin B, or  P. aeruginosa  Exotoxin A.