Yeast membrane protein expression system and its application in drug screening

Abstract

The invention relates to an in vitro cell based expression system for overexpressing heterologous pump proteins associated with drug resistance into the membrane of the host cell for drug screening applications.

1 . A protein expression system comprising: 
 i) a host yeast cell; and    ii) a vector comprising the coding sequence of a target heterologous membrane protein, said sequence being under the control of a promoter which, upon transformation of said host cell and chromosomal integration, causes over-expression of the functional target protein in the membrane of the host cell.    
     
     
         2 . A protein expression system as claimed in  claim 1 , wherein the host cell is a yeast cell of the genus  Saccharomyces.    
     
     
         3 . A protein expression system as claimed in  claim 1  or  2 , wherein the host cell comprises a mutant strain deficient in one or more naturally occurring membrane proteins thereby enabling the target protein to be expressed prominently in the membrane of the host cell and to be accessible for drug screening applications.  
     
     
         4 . A protein expression system as claimed in  claim 3 , wherein the host cell is deficient in drug efflux pump proteins.  
     
     
         5 . A protein expression system as claimed in any one of  claims 2  to  4 , wherein the host cell is the  Saccharomyces cerevisiae  AD1-8u −  strain.  
     
     
         6 . A protein expression system as claimed in any preceding claim, wherein the host cell contains a mutation that leads to the formation of secretory vesicles whose ability to fuse normally with the plasma membrane is temperature sensitive.  
     
     
         7 . A protein expression system as claimed in  claim 6 , wherein the host cell is a sec6-4 mutant of the AD1-8u −  strain.  
     
     
         8 . A protein expression system as claimed in  claim 1 , wherein the coding sequence of the target protein is incorporated into the host cell in a defined location in the genome.  
     
     
         9 . A protein expression system as claimed in  claim 1 , wherein the coding sequence comprises the entire natural coding sequence of the heterologous target protein, or a functional fragment or variant thereof.  
     
     
         10 . A protein expression system as claimed in  claim 8  or  9 , where the target heterologous membrane protein is a drug efflux pump protein selected from the group consisting of pump proteins involved in multidrug resistance in fungi, P-glycoprotein, cystic fibrosis transmembrane conductance regulator and any other human, animal, plant or microbial plasma membrane proteins that play a role in conferral of drug resistance.  
     
     
         11 . A protein expression system as claimed in any one of  claims 3  to  10  wherein the target heterologous membrane protein is of the same class of membrane proteins which have been deleted from the host cell.  
     
     
         12 . A protein expression system as claimed in  claim 11 , where the target heterologous membrane proteins are selected from  Candida albicans  Cdr1p, Cdr2p and from  Candida galbrata  Cdr1p and Pdh1p.  
     
     
         13 . A protein expression system as claimed in any one of  claims 3  to  10  wherein the target heterologous membrane protein is of a different class to the class of membrane proteins which have been deleted from the host cell.  
     
     
         14 . A protein expression system as claimed in  claim 13  wherein the target heterologous membrane product is selected from the  Candida albicans  Ben R P and Erg11p.  
     
     
         15 . A protein expression system as claimed in  claim 1 , wherein the vector is a plasmid vector.  
     
     
         16 . A protein expression system as claimed in  claim 15 , wherein the plasmid vector contains elements which allow replication in  Escherichia coli.    
     
     
         17 . A protein expression system as claimed in  claim 15 , wherein the plasmid vector is pABC.  
     
     
         18 . A protein expression system as claimed in  claim 1 , wherein the promoter is a  Saccharomyces cerevisiae  promoter.  
     
     
         19 . A protein expression system as claimed in  claim 18 , wherein the promoter is selected from the group comprising  S. cerevisiae  PDR5, PMA1, CTR3, ADH1, PGK and GAL and bacterial tet0 promoters and tet0::ScHOP1 controllable cassette.  
     
     
         20 . A protein expression system as claimed in  claim 19 , wherein the promoter is the PDR5 promoter.  
     
     
         21 . A protein expression system as claimed in any one of  claims 18  to  20 , wherein the  Saccharomyces  promoter is under the control of a transcriptional regulator so as to induce over-expression of the target protein coding sequence in the membrane of the host cell.  
     
     
         22 . A protein expression system as claimed in  claim 21 , wherein the transcriptional regulator is the Pdr1-3p transcriptional regulator.  
     
     
         23 . A method of screening for drugs useful as a pharmaceutical or agrochemical comprising the steps of: 
 i) transforming the chromosomal DNA of a host yeast cell with DNA comprising the coding sequence of a target heterologous membrane protein, said sequence being under the control of a host promoter leading to over-expression of the functional target protein in the membrane of the host cell;    ii) introducing at least one candidate compound to said host cell environment or the environment of a plasma membrane fraction derived from the transformed host strain; and    iii) measuring the effect, if any, of the candidate compound on the host cell growth and/or viability and/or specific biochemical or physiological functions mediated by the target membrane protein; and/or measuring the binding of the candidate compound to the target membrane protein.    
     
     
         24 . A method as claimed in  claim 23 , wherein the host cell is a yeast cell of the genus  Saccharomyces.    
     
     
         25 . A method as claimed in  claim 24 , wherein the host cell is a  Saccharomyces  cell which has been genetically altered to be depleted in one or more natural membrane proteins.  
     
     
         26 . A method as claimed in  claim 25 , wherein the host cell is the  Saccharomyces cerevisiae  AD 1-8u −  strain.  
     
     
         27 . A method as claimed in  claim 25  or  26 , wherein the host cell is a sec6-4 mutant of the AD1-8u −  strain.  
     
     
         28 . A method as claimed in  claim 23 , wherein the target heterologous membrane protein comprises a drug efflux pump protein.  
     
     
         29 . A method as claimed in  claim 23 , wherein the drug efflux drug pump protein is selected from the group consisting of pump proteins involved in multidrug resistance in fungi, the P-glycoprotein, the cystic fibrosis transmembrane conductance regulator and other human, animal, plant and microbial plasma membrane proteins that play a role in the conferral of drug resistance.  
     
     
         30 . A method as claimed in any one of  claims 23  to  29 , wherein the target membrane protein is a drug efflux pump protein and the candidate compound is an efflux pump inhibitor.  
     
     
         31 . A method as claimed in  claim 23 , wherein said coding sequence and promoter are introduced into said host cell via a plasmid vector.  
     
     
         32 . A method as claimed in  claim 31 , wherein the plasmid vector contains elements which allow replication in  Escherichia coli.    
     
     
         33 . A method as claimed in  claim 31 , wherein the plasmid vector is pABC3.  
     
     
         34 . A method as claimed in any one of  claims 31  to  33 , wherein said promoter is a  Saccharomyces cerevisiae  promoter.  
     
     
         35 . A method as claimed in  claim 34 , wherein the promoter is selected from the group consisting of  S. cerevisiae  PDR5, PMA1, CTR3, ADH1, PGK and GAL and bacterial tet0 promoters and the tet0::ScHOP1 controllable cassette.  
     
     
         36 . A method as claimed in  claim 34  or  35 , wherein the promoter is the PDR5 promoter.  
     
     
         37 . A method as claimed in any one of  claims 34  to  36 , wherein  Saccharomyces  promoter is under the control of a transcriptional regulator so as to induce over-expression of the target protein coding sequence in the membranes of the host cell.  
     
     
         38 . A method as claimed in  claim 37 , wherein the transcriptional regulator is the Pdr1-3p transcriptional regulator.  
     
     
         39 . A vector suitable for use in the overexpression of a target heterologous membrane protein in a yeast host cell comprising pABC3.  
     
     
         40 . A bioactive, pharmaceutical or agrochemical compound identified using the method of any one of  claims 23  to  38  or the protein expression system of any one of  claims 1  to  22 .  
     
     
         41 . A compound as claimed in  claim 40 , wherein said compound was obtained from compound libraries.  
     
     
         42 . A compound as claimed in  claim 40  or  41 , comprising KN20 as defined herein.  
     
     
         43 . A compound comprising KN20 as defined herein suitable for use as an antifungal.  
     
     
         44 . An antifungal composition comprising compound KN20 as defined herein together with a suitable carrier or diluent.  
     
     
         45 . An antifungal composition comprising compound KN20 as defined herein in a mixture with a known antifungal compound selected from the group consisting of fluconazole and other xenobiotics that are transported by multidrug efflux mechanisms.  
     
     
         46 . A purified membrane protein produced by the method of any one of  claims 23  to  38  or protein expression system of any one of  claims 1  to  22 .  
     
     
         47 . A kit for screening for drugs useful as a pharmaceutical or agrochemical comprising: 
 (i) a host cell;    (ii) a vector containing the coding sequence of a target heterologous membrane protein, said sequence being under the control of a promoter which, upon transformation of said host cell and chromosomal integration, causes over-expression of the functional target protein in the membrane of the host cell; and    (iii) instructions to carry out said transformation and drug screening procedures.    
     
     
         48 . A kit as claimed in  claim 47  wherein said host cell comprises  S. crevisiae  AD1-8u −  and said vector is pABC3 and comprises a PDR5 promoter and a coding sequence of a heterologous drug efflux pump protein.  
     
     
         49 . A kit as claimed in  claim 47  or  48 , wherein said pump protein is selected from the group comprising  C. albicans  Cdr1p, Cdr2p, Ben R p, Erg11p and  C. galbrata  Cdr1p and Pdh1p.