Single chain class I major histo-compatibility complexes, constructs encoding same and methods of generating same

Abstract

Provided are methods of generating a functional mammalian single chain MHC class I complex in prokaryotic expression systems and a host cell transformed with expression construct(s) capable of expressing a functional human single chain MHC class I complex capable of presenting specific antigenic peptides restricted to specific CTL clones.

What is claimed is:  
     
         1 . A host cell being co-transformed with: 
 (a) a first expression construct including a first polynucleotide encoding a functional mammalian β-2 microglobulin, being translationally fused upstream of a second polynucleotide encoding a functional MHC class I heavy chain; and    (b) a second expression construct including a third polynucleotide encoding an antigenic peptide, wherein when said first, second and third polynucleotides are co-expressed in the host cell, an MHC class I-antigenic peptide complex is formed.    
     
     
         2 . The host cell of  claim 1 , wherein said first expression construct further includes an in-frame linker polynucleotide sequence encoding a linker peptide interposed between said first and said second polynucleotides.  
     
     
         3 . The host cell of  claim 1 , wherein said cell is a eukaryotic cell.  
     
     
         4 . The host cell of  claim 1 , wherein said cell is a bacterial cell.  
     
     
         5 . A method of producing a functional MHC class I molecule comprising the steps of: 
 (a) expressing, in bacteria, a single chain MHC class I polypeptide including a functional mammalian β-2 microglobulin amino acid sequence covalently linked to a functional mammalian MHC class I heavy chain amino acid sequence; and    (b) isolating said single chain MHC class I polypeptide.    
     
     
         6 . The method of  claim 5 , further comprising the step of: 
 (c) refolding said single chain MHC class I polypeptide in presence of an antigenic peptide capable of binding said single chain MHC class I polypeptide, to thereby generate an MHC class I-antigenic peptide complex.    
     
     
         7 . The method of  claim 5 , further comprising the step of: 
 (d) isolating said MHC class I-antigenic peptide complex via size exclusion chromatography.    
     
     
         8 . The method of  claim 5 , wherein said antigenic peptide is co-expressed along with said single chain MHC class I polypeptide in said bacteria.  
     
     
         9 . The method of  claim 5 , wherein step (a) is effected such that said single chain MHC class I polypeptide forms inclusion bodies in said bacteria.  
     
     
         10 . The method of  claim 8 , wherein said antigenic peptide and said single chain MHC class I polypeptide form inclusion bodies in said bacteria.  
     
     
         11 . The method of  claim 9 , wherein said step of isolating said polypeptide further includes the steps of: 
 (i) denaturing said inclusion bodies so as to release protein molecules therefrom; and    (ii) renaturing said protein molecules.    
     
     
         12 . The method of  claim 11 , wherein said step of renaturing said protein molecules is effected in the presence of an antigenic peptide capable of binding said single chain MHC class I polypeptide.  
     
     
         13 . The method of  claim 12 , wherein said antigenic peptide is co-expressed along with said single chain MHC class I polypeptide in said bacteria.  
     
     
         14 . The method of  claim 5 , wherein said mammalian β-2 microglobulin amino acid sequence is a human β-2 microglobulin amino acid sequence and further wherein said mammalian MHC class I heavy chain amino acid sequence is a human MHC class I heavy chain amino acid sequence.