Expression of heterologous multi-domain proteins in yeast

Abstract

This invention demonstrates the utility of a yeast expression system for the expression of functional heterologous multi-domain proteins in yeast. The yeast expression system allows for the inclusion of a plurality of (up to three) modular expression cassettes which may encode multiple polypeptide chains of a heterologous multi-domain protein on a single plasmid (Twin Cassette). Because multiple polypeptide chains may be encoded for by the expression cassettes of the present invention in a single vector, the system can produce equivalent amounts of the multiple polypeptide chains, thereby enhancing the yield of a functional heterologous multi-domain protein. For example, functional monoclonal antibodies (MAbs) comprising a heavy chain and a light chain of an immunoglobulin (IgG), and functional immunotoxins comprising an antibody domain and an oxidase toxin may be produced using the Yeast expression system of the present invention. In addition, functional single chain antibodies, antibody fragments and chimeric antibodies may also be produced.

1 . A vector including one or more expression cassettes wherein one of said expression cassettes comprises a nucleic acid encoding a heterologous recombinant fusion protein which comprises an immunological molecule and a toxin.  
     
     
         2 . The vector of  claim 1  wherein the expression cassettes further comprise promoter sequences operatively linked to the nucleic acid encoding the heterologous recombinant fusion protein.  
     
     
         3 . The vector of  claim 2  wherein the expression cassettes further comprise translation initiation sequences and wherein said promoter sequences are located flush with and 5′ to said translation initiation sequences.  
     
     
         4 . The vector of  claim 1  wherein the expression cassettes further comprise a nucleic acid encoding a secretory signal sequence.  
     
     
         5 . The vector of  claim 4  wherein the secretory signal sequence is selected from the group consisting of human secretory signal sequences, yeast secretory sequences and secretory sequences which are native to the heterologous fusion protein.  
     
     
         6 . (canceled)  
     
     
         7 . (canceled)  
     
     
         8 . (canceled)  
     
     
         9 . (canceled)  
     
     
         10 . The vector of  claim 1  further including an expression cassette comprising a nucleic acid encoding an accessory molecule.  
     
     
         11 . The vector of  claim 10  wherein the accessory molecule is a chaperone protein.  
     
     
         12 . The vector of  claim 11  wherein the chaperone protein is selected from the group consisting of the BiP and protein disulfide isomerase (PDI).  
     
     
         13 . A method of producing a functional heterologous recombinant multichain or single-chain fusion protein comprising: 
 (a) transforming a  Saccharomyces  yeast host cell with a vector, wherein the vector comprises multiple expression cassettes, and wherein at least one of the expression cassettes encodes the fusion protein;    (b) growing the transformed cell in culture to a stage of log phase growth; and    (c) inducing the cell culture to express the heterologous recombinant multichain or single-chain fusion protein, wherein the fusion protein comprises an immunotoxin, and wherein the immunotoxin comprises an antibody domain and an oxidase toxin domain.    
     
     
         14 . The method of  claim 13  wherein the yeast cell is  Saccharomyces cerevisiae.    
     
     
         15 . The method of  claim 14  wherein the strain of  S. cerevisiae  is selected from the group consisting of Y112, Y113, Y114, Y115, Y116, Y117, Y118, Y119, Y120, Y121, Y122, Y123, Y124 and Y125.  
     
     
         16 . The method of  claim 13  wherein the yeast host cell comprises one or more mutations in glycosylation pathways.  
     
     
         17 . The method of  claim 16  wherein the recombinant fusion protein is glycosylated.  
     
     
         18 . The method of  claim 13  wherein the yeast host cell comprises supersecretory activity.  
     
     
         19 . (canceled)  
     
     
         20 . The method of  claim 13  wherein the transformed yeast cell is grown under fermentation parameters that improve the production of the recombinant fusion protein.  
     
     
         21 . The method of  claim 20  wherein the fermentation parameters are selected from the group consisting of carbon sources, buffering systems, media formulations, vitamin levels, trace salt levels, temperature, aeration levels, oxygen levels, pH, induction time, and length of induction.  
     
     
         22 . (canceled)  
     
     
         23 . The method of  claim 13  wherein the antibody domain comprises two chains and wherein one of the chains is operably linked to the toxin domain.  
     
     
         24 . The method of  claim 23  wherein one of the two chains is a heavy chain, and wherein the other chain is a light chain.  
     
     
         25 . The method of  claim 23  wherein the vector comprises multiple expression cassettes.  
     
     
         26 . The method of  claim 13  wherein the toxin is selected from the group consisting of glucose oxidase, glucose-oxidase-related toxin, horseradish peroxidase, and horseradish peroxidase-related toxin.  
     
     
         27 . A vector comprising at least two expression cassettes, wherein a first expression cassette comprises a nucleic acid encoding a heterologous recombinant fusion protein, wherein the fusion protein comprises an immunological molecule and a toxin, wherein a second expression cassette comprises a nucleic acid encoding an accessory molecule, and wherein the accessory molecule is a chaperone protein selected from the group consisting of a BiP and a protein disulfide isomerase (PDI).  
     
     
         28 . The vector of  claim 27 , wherein a third expression cassette comprises a nucleic acid encoding an accessory molecule, and wherein the accessory molecule is a chaperone protein selected from the group consisting of a BiP and a protein disulfide isomerase (PDI).