Recombinant prion-like genes and proteins and materials and methods comprising the same

Abstract

The present invention provides novel polypeptides comprising a prion-aggregation domain and a second domain; novel polynucleotides encoding such polypeptides; host cells transformed or transfected with such polynucleotides; and methods of making and using the foregoing.

1 . A polynucleotide comprising a nucleotide sequence that encodes a chimeric polypeptide, said polynucleotide comprising: 
 a nucleotide sequence encoding at least one SCHAG amino acid sequence fused in frame with a nucleotide sequence encoding at least one polypeptide of interest other than a marker protein, a glutathione S-transferase (GST) protein, or a Staphylococcal nuclear protein.    
     
     
         2 . A polynucleotide according to  claim 1  wherein the at least one SCHAG amino acid sequence comprises at least one prion-aggregation domain of a prion protein.  
     
     
         3 . A polynucleotide according to  claim 2 , further comprising a nucleotide sequence encoding a translation initiation codon and a secretory signal peptide fused in frame and upstream of the encoding sequences.  
     
     
         4 . A polynucleotide according to  claim 2 , further comprising a translation initiation codon fused in frame and upstream (5′) of the encoding sequences, and a translation stop codon fused in frame and downstream (3′) of the encoding sequences.  
     
     
         5 . A polynucleotide according to  claim 4  wherein said polynucleotide further includes a sequence encoding an endopeptidase or chemical recognition sequence fused in frame between the sequence encoding the at least one prion-aggregation domain and the sequence encoding the polypeptide of interest.  
     
     
         6 . A polynucleotide according to  claim 4  wherein the nucleotide sequence encoding the at least one prion-aggregation domain is fused upstream (5′) of the sequence encoding the at least one polypeptide of interest.  
     
     
         7 . A polynucleotide according to  claim 4  further comprising a promoter sequence operatively connected upstream (5′) of the encoding sequences.  
     
     
         8 . A polynucleotide according to  claim 7  further comprising a polyadenylation signal sequence operatively connected downstream (3′) of the encoding sequences.  
     
     
         9 . A polynucleotide according to  claim 4 , wherein the polynucleotide further includes a sequence encoding a selectable marker protein.  
     
     
         10 . A polynucleotide according to  claim 4 , wherein the at least one prion-aggregation domain comprises the prion aggregation domain of a protein selected from the group consisting of: mammalian prion proteins (PrP) and Ht proteins; Sup35 proteins; Ure2 proteins; and Rnq1 proteins.  
     
     
         11 . A polynucleotide according to  claim 4  wherein the at least one prion-aggregation domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 46, 47, and 50 and prion aggregation domain fragments thereof.  
     
     
         12 . A polynucleotide according to  claim 4 , wherein the at least one prion-aggregation domain comprises the amino acid sequence of positions 2-113 of SEQ ID NO: 2.  
     
     
         13 . A polynucleotide according to  claim 4 , wherein the at least one prion-aggregation domain comprises the amino acid sequence of positions 2-65 of SEQ ID NO: 4.  
     
     
         14 . A polynucleotide according to  claim 4  wherein the at least one polypeptide of interest is an enzyme.  
     
     
         15 . A polynucleotide according to  claim 4  wherein the at least one polypeptide of interest is a polypeptide capable of binding a composition of interest.  
     
     
         16 . A polynucleotide according to  claim 4  wherein the at least one polypeptide of interest comprises at least one antigen binding domain of an antibody.  
     
     
         17 . A polynucleotide according to  claim 4  wherein the at least one polypeptide of interest comprises at least one ligand binding domain of a ligand binding protein.  
     
     
         18 . A polynucleotide according to  claim 4 , wherein the at least one polypeptide of interest comprises a ligand of a cell surface receptor.  
     
     
         19 . A host cell transformed or transfected with a polynucleotide according to  claim 4 .  
     
     
         20 . A vector comprising a polynucleotide according to  claim 4 .  
     
     
         21 . A host cell transformed or transfected with a vector according to  claim 20 .  
     
     
         22 . A polynucleotide comprising a nucleotide sequence that encodes a chimeric polypeptide, said chimeric polypeptide comprising an amyloidogenic domain that causes the polypeptide to aggregate with identical polypeptides into fibrils, fused to a domain comprising a polypeptide of interest; 
 wherein the amyloidogenic domain comprises an amyloidogenic amino acid sequence of a naturally occurring protein and further includes a duplication of at least a portion of said naturally occurring amyloidogenic amino acid sequence, said duplication increasing the amyloidogenic affinity of said chimeric polypeptide relative to an identical chimeric polypeptide lacking said duplication.    
     
     
         23 . A polynucleotide according to  claim 22  wherein the naturally occurring protein comprises a Sup35 protein of  Saccharomyces cereviciae  characterized by the partial amino acid sequence PQGGYQQYN, and wherein said duplication includes the amino acid sequence PQGGYQQYN.  
     
     
         24 . A polynucleotide comprising a nucleotide sequence that encodes a chimeric polypeptide, said chimeric polypeptide comprising an amyloidogenic domain that causes the polypeptide to aggregate with identical polypeptides into fibrils, fused to a domain comprising a polypeptide of interest; wherein the amyloidogenic domain comprises amyloidogenic amino acid sequences of at least two naturally occurring amyloidogenic proteins.  
     
     
         25 . A polynucleotide encoding a chimeric polypeptide, said polypeptide comprising at least two prion-aggregation domains fused in frame with at least one polypeptide of interest.  
     
     
         26 . A chimeric polypeptide encoded by a polynucleotide of  claim 1 .  
     
     
         27 . A composition comprising a purified polypeptide according to  claim 26 .  
     
     
         28 . A chimeric polypeptide encoded by a polynucleotide of  claim 22 .  
     
     
         29 . A chimeric polypeptide encoded by a polynucleotide of  claim 24 .  
     
     
         30 . A chimeric polypeptide encoded by a polynucleotide of  claim 25 .  
     
     
         31 . A fibril comprising an ordered aggregate of chimeric polypeptides according to  claim 26 .  
     
     
         32 . A composition comprising at least one polypeptide aggregate, said polypeptide aggregate comprising a plurality of chimeric polypeptides according to  claim 26 .  
     
     
         33 . A composition according to  claim 32  wherein said polypeptide aggregate is insoluble in water.  
     
     
         34 . A method comprising the steps of: 
 transforming or transfecting a cell with a polynucleotide according to  claim 1;  and    growing the cell under conditions which result in expression of said chimeric polypeptide in said cell.    
     
     
         35 . A method according to  claim 34 , further comprising the step of isolating the chimeric polypeptide from the cell or from growth medium of the cell.  
     
     
         36 . A method according to  claim 35 , further comprising the step of proteolyically detaching the SCHAG amino acid sequence of the protein from the polypeptide of interest.  
     
     
         37 . A method according to  claim 36 , further comprising the step of isolating the protein of interest from the SCHAG amino acid sequence.  
     
     
         38 . A method of making a protein of interest, comprising the steps of: 
 transforming or transfecting a cell with a polynucleotide, said polynucleotide comprising a nucleotide sequence that encodes a chimeric polypeptide, said chimeric polypeptide comprising an amyloidogenic domain that causes the polypeptide to aggregate with identical polypeptides into fibrils, fused to domain comprising a polypeptide of interest;    growing the cell under conditions which result in expression of said chimeric polypeptide in said cell and aggregation of said chimeric polypeptide into fibrils; and    isolating the chimeric polypeptide from the cell or from growth medium of the cell.    
     
     
         39 . A method according to  claim 38  wherein said isolating step comprises the step of separating the fibrils from soluble proteins of said cell.  
     
     
         40 . A method according to  claim 39 , further comprising the steps of proteolytically detaching the amyloidogenic domain of the chimeric protein from the polypeptide of interest; and isolating the polypeptide of interest.  
     
     
         41 .- 48 . (canceled)  
     
     
         49 . A composition comprising an ordered aggregate of at least two chimeric polypeptides according to  claim 1 , said at least two chimeric polypeptides having compatible SCHAG amino acid sequences and distinct polypeptides of interest.  
     
     
         50 . A composition according to  claim 49  wherein the at least two chimeric polypeptides comprise identical SCHAG amino acid sequences.  
     
     
         51 . A composition according to  claim 49  wherein the ordered aggregate comprises a fiber and wherein the polypeptides of interest retain native biological activity.  
     
     
         52 . A host cell transformed or transfected with at least two polynucleotides according to  claim 1 , wherein said two polynucleotides comprise compatible SCHAG amino acid sequences and distinct polypeptides of interest.  
     
     
         53 . A cell culture comprising cells transformed or transfected with a polynucleotide according to  claim 1 , wherein the cells express the chimeric polypeptide encoded by the polynucleotide, and wherein the cell culture includes cells wherein said chimeric polypeptide is present in an aggregated state and cells free of aggregated chimeric polypeptide.  
     
     
         54 . A cell culture according to  claim 53 , wherein at least some cells convert between a phenotype characterized by aggregated chimeric polypeptide and a phenotype characterized by both the presence of unaggregated chimeric polypeptide and the absence of aggregated chimeric polypeptide.  
     
     
         55 .- 92 . (canceled)  
     
     
         93 . A composition comprising a fibril according to  claim 31  attached to a solid support.  
     
     
         94 . A composition comprising an ordered aggregate according to  claim 49  attached to a solid support.  
     
     
         95 .- 100 . (canceled)