Polypeptide factor from a thermophilic eubacterial species and use thereof in the production of functional, heterologous proteins in an expression host

Abstract

A polypeptide factor derived from the thermophilic eubacterial species Thermus thermophilus has universal protein expression-assisting activity. The polypeptide factor has been named the CzrB protein active in full length or truncated form has the potential to act as a universal protein expression-assisting molecule which can increase the yields of all heterologous proteins produced in E. coli by a mechanism that is independent of the protein being expressed.

1 . A polypeptide factor derived from a thermophilic eubacterial species, said polypeptide factor having universal protein expression-assisting activity.  
     
     
         2 . The polypeptide of  claim 1 , wherein the thermophilic eubacterial species is  Thermus thermophilus.    
     
     
         3 . The polypeptide factor of  claim 1  or  2 , having an amino acid sequence defined as amino acid position 1 to amino acid position 291 in FIG. 2 (SEQ ID NO:1).  
     
     
         4 . The polypeptide factor of  claim 3 , having SEQ ID NO:2.  
     
     
         5 . An isolated DNA sequence encoding the polypeptide factor of  claim 1 .  
     
     
         6 . An isolated DNA sequence encoding the polypeptide factor of  claim 2 .  
     
     
         7 . An isolated DNA sequence encoding the polypeptide factor of  claim 3  and having SEQ ID NO:3.  
     
     
         8 . An isolated DNA sequence encoding the polypeptide factor of  claim 4  and having SEQ ID NO:4.  
     
     
         9 . An isolated DNA sequence comprising the structural gene encoding the polypeptide factor of  claim 3  and a flanking sequence containing a control element for the expression of said polypeptide factor.  
     
     
         10 . An isolated DNA sequence according to  claim 9  having SEQ ID NO:5.  
     
     
         11 . A method for increasing production of heterologous proteins in a bacterial host cell, which comprises contacting said bacterial host cell with an effective amount of the polypeptide factor of  claim 1  during the expression of said heterologous protein.  
     
     
         12 . A method according to  claim 11 , wherein the bacterial host cell is an  E. coli  host cell.  
     
     
         13 . A vector comprising the isolated DNA sequence of  claim 5 .  
     
     
         14 . A host cell containing the isolated DNA sequence of  claim 5 .  
     
     
         15 . A method for increasing production of heterologous protein in a bacterial host cell, which method comprises cultivating said host cell under conditions permitting expression of said DNA sequence of  claim 5  or  9 .  
     
     
         16 . A method according to  claim 15 , wherein the heterologous protein is a eucaryotic protein.  
     
     
         17 . A method for the production of heterologous functional protein in an  E. coli  host cell, said method comprising co-cultivating DNA for said heterologous functional protein with the DNA sequence of  claim 5  or  9 .  
     
     
         18 . A method for the production of heterologous function protein in an  E. coli  host cell, which method comprises co-expression of said polypeptide factor of  claim 1 .  
     
     
         19 . A method of reducing stress in an expressing bacterial cell, which method comprises co-expressing a heterologous protein and the polypeptide factor of  claim 1 .  
     
     
         20 . A method of optimising expression of a heterologous protein in an expressing bacterial cell, which method comprises co-expressing the heterologous protein and the polypeptide factor of  claim 1 .  
     
     
         21 . An antibody to a polypeptide factor as defined in  claim 1 .  
     
     
         22 . A method of purifying a protein with universal protein expression-assisting activity, said method comprising contacting a cell extract with an antibody of  claim 21 .  
     
     
         23 . The polypeptide factor of  claim 1 , which has homology with metal ion efflux proteins from other eucaryotic species.  
     
     
         24 . The polypeptide factor of  claim 1 , which confers on  E. coli  increased resistance to heavy metal ions.