Assembly and screening of highly complex and fully human antibody repertoire in yeast

Abstract

Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently vary within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced. The library of expression vectors can be efficiently generated in yeast cells through homologous recombination; and the encoded proteins complexes with high binding affinity to their target molecule can be selected by high throughput screening in vivo or in vitro.

What is claimed is:  
     
         1 . A method for selecting tester protein complexes capable of binding to a target peptide or protein, the method comprising: 
 expressing a library of tester protein complexes in yeast cells, each tester protein complex being formed between a first polypeptide subunit whose sequence varies within the library and a second polypeptide subunit which is expressed as a separate protein from the first polypeptide subunit and whose sequence varies within the library independently of the first polypeptide;    expressing a target fusion protein in the yeast cells expressing the tester protein complexes, the target fusion protein comprising a target peptide or protein; and    selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester protein complex to the target fusion protein.    
     
     
         2 . The method of  claim 1 , wherein expressing the library of tester protein complexes includes 
 transforming a library of tester expression vectors into the yeast cells which contain a reporter construct comprising the reporter gene whose expression is under transcriptional control of a transcription activator comprising an activation domain and a DNA binding domain, each tester expression vector comprising 
 a first transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator,  
 a first nucleotide sequence encoding the first polypeptide subunit fused which is expressed as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator, and  
 a second nucleotide sequence encoding the second polypeptide subunit which is expressed as a separate protein from the first polypeptide subunit.  
   
     
     
         3 . The method of  claim 2 , wherein expressing a target fusion protein includes 
 transforming a target expression vector into the yeast cells simultaneously or sequentially with the library of tester expression vectors, the target expression vector comprising 
 a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors; and  
 a target sequence encoding the target protein or peptide; and  
   expressing the target fusion protein from the target expression vector.    
     
     
         4 . The method of  claim 1 , wherein the steps of expressing the library of tester protein complexes and expressing the target fusion protein include causing mating between first and second populations of haploid yeast cells of opposite mating types,  
       wherein 
 the first population of haploid yeast cells comprises 
 a library of tester expression vectors for the library of tester fusion proteins, each tester expression vector comprising 
 a first transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator,  
 a first nucleotide sequence encoding the first polypeptide subunit fused which is expression as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator, and  
 a second nucleotide sequence encoding the second polypeptide subunit which is expressed as a separate protein from the first polypeptide subunit; and  
 
 
 the second population of haploid yeast cells comprises a target expression vector comprising 
 a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and  
 a target sequence encoding the target protein or peptide; and  
 
 either the first or second population of haploid yeast cells comprises a reporter construct comprising the reporter gene whose expression is under transcriptional control of the transcription activator.  
 
     
     
         5 . The method of  claim 4 , wherein the haploid yeast cells of opposite mating types are α and a type strains of yeast.  
     
     
         6 . The method of  claim 5 , wherein the mating between the first and second populations of haploid yeast cells of α and a type strains is in a rich nutritional culture medium.  
     
     
         7 . The method of  claim 1 , wherein the diversity of the protein complexes encoded by the library of yeast expression vectors is at least 1×10 7 .  
     
     
         8 . The method of  claim 1 , wherein the diversity of the protein complexes encoded by the library of yeast expression vectors is at least 1×10 10 .  
     
     
         9 . The method of  claim 1 , wherein the diversity of the protein complexes encoded by the library of yeast expression vectors is at least 1×10 12 .  
     
     
         10 . The method of  claim 1 , wherein the first nucleotide sequence in the library of expression vectors comprises a coding sequence of an antibody light-chain region, and the second nucleotide sequence comprises a coding sequence of an antibody heavy-chain region.  
     
     
         11 . The method of  claim 1 , wherein the conformation of the protein complexes expressed by the library of expression vectors mimics a conformation of an antibody.  
     
     
         12 . The method of  claim 1 , further comprising: 
 isolating the tester expression vector from the selected clones; and    mutagenizing the first and second nucleotide sequences in the isolated tester expression vectors to form a library of mutagenized expression vectors.    
     
     
         13 . The method of  claim 12 , wherein the mutagenesis is selected from the group consisting of error-prone PCR mutagenesis, site-directed mutagenesis, DNA shuffling and combinations thereof.  
     
     
         14 . The method of  claim 1 , wherein the target fusion protein comprises an antigen associated with a disease state.  
     
     
         15 . The method of  claim 1 , wherein the target fusion protein comprises a tumor-surface antigen.  
     
     
         16 . The method of  claim 1 , wherein the target fusion protein comprises a human growth factor receptor.  
     
     
         17 . The method of  claim 16 , wherein the human growth factor is selected from the group consisting of epidermal growth factors, transferrin, insulin-like growth factor, transforming growth factors, interleukin-1, and interleukin-2.  
     
     
         18 . The method of  claim 1 , wherein the protein encoded by the reporter gene is selected from the group consisting of β-galactosidase, α-galactosidase, luciferase, β-glucuronidase, chloramphenicol acetyl transferase, secreted embryonic alkaline phosphatase, green fluorescent protein, enhanced blue fluorescent protein, enhanced yellow fluorescent protein, and enhanced cyan fluorescent protein.  
     
     
         19 . A method for selecting tester proteins capable of binding to a target peptide or protein, the method comprising: 
 expressing a library of tester protein complexes in yeast cells, each tester protein complex being formed in vivo between a first polypeptide subunit whose sequence varies within the library and a second polypeptide subunit which is expressed as a separate protein from the first polypeptide subunit and whose sequence varies within the library independently of the first polypeptide;    expressing a plurality of target fusion proteins in the yeast cells expressing the tester proteins, each of the target fusion proteins comprising a target peptide or protein; and    selecting those yeast cells in which a reporter gene is expressed, the expression of the reporter gene being activated by binding of the tester fusion to the target fusion protein.    
     
     
         20 . The method of  claim 19 , wherein the steps of expressing the library of tester protein complexes and expressing the plurality of the target fusion proteins includes causing mating between first and second populations of haploid yeast cells of opposite mating types,  
       wherein 
 the first population of haploid yeast cells comprises 
 a library of tester expression vectors for the library of tester fusion proteins, each tester expression vector comprising 
 a first transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator,  
 a first nucleotide sequence encoding the first polypeptide subunit fused which is expression as a fusion protein with either the activation domain or the DNA binding domain of the transcription activator, and  
 a second nucleotide sequence encoding the second polypeptide subunit which is expressed as a separate protein from the first polypeptide subunit; and  
 
 
 the second population of haploid yeast cells comprises a plurality of target expression vectors, each of the target expression vector comprising 
 a second transcription sequence encoding either the activation domain or the DNA binding domain of the transcription activator which is not expressed by the library of tester expression vectors, and  
 a target sequence encoding the target protein or peptide,  
 
 wherein either the first or second population of haploid yeast cells further comprises a reporter construct comprising the reporter gene whose expression is under transcriptional control of the transcription activator.  
 
     
     
         21 . The method of  claim 20 , wherein members of the library of tester expression vectors are arrayed as individual yeast clones in one or more multiple-well plates.  
     
     
         22 . The method of  claim 20 , wherein members of the library of target expression vectors are arrayed as individual yeast clones in one or more multiple-well plates.  
     
     
         23 . The method of  claim 20 , wherein the mating is based on clonal mating in which each yeast clone containing members of the tester expression vectors is mated individually with each of the members of the library of target expression vector.