Isolating biological modulators from biodiverse gene fragment libraries

Abstract

The present invention provides a method for identifying a modulator or mediator of a biological activity, which activity includes antigenicity and or immunogenicity, said method comprising the step of: (i) producing a gene fragment expression library derived from defined nucleotide sequence fragments; and (ii) assaying the expression library for at least an amino acid sequence derived from step (i) for a biological activity wherein that activity is different from any activity the amino acid sequence may have in its native environment.

1 - 23 . (canceled)  
     
     
         24 . A method of screening a genome fragment expression library comprising the steps of: 
 (i) producing a gene fragment expression library expressing a plurality of different nucleotide sequences from different organisms, wherein said library is made from defined nucleotide sequence fragments from a sequenced genome of a micro-organism and/or a sequenced compact genome of a eukaryotic species; and    (ii) assaying the gene fragment expression library to identify a peptide that has a biological activity wherein said biological activity is characterised as being different from any activity the peptide has in its native environment.    
     
     
         25 . The method of  claim 24  wherein the nucleic acid fragments are from an organism selected from the group consisting of  Fugu rubripes, Caenorhabditis elegans, Saccharomyces cerevisiae, Escherichia coli, Aquifex aeliticus, Methanococcus jannaschii, Bacillus subtilis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningiditus, Synechocystis  sp.,  Bordetella pertussis, Pasteurella multocida, Pseudomonas aeruginosa, Borrelia burgdorferi, Menthbacterium thermoautotrophicum, Mycoplasma pneumoniae, Archaeoglobus fulgidis , and  Vibrio harveyi.    
     
     
         26 . The method of  claim 24  wherein each of the genomes used in the method are provided in equal molar amounts to ensure that an equal proportion of the nucleic acid fragments are included in the method.  
     
     
         27 . The method of  claim 24  wherein the peptide encoded by the nucleic acid fragment comprises a sequence of amino acids of at least about 1 to 100 amino acids in length such as about 30 amino acids in length  
     
     
         28 . The method of  claim 24  wherein the complexity of the gene fragment expression library is augmented by subjecting the nucleic acid fragments to methods that mis-read or mutate the sequence.  
     
     
         29 . The method of  claim 28  wherein the complexity of the gene fragment expression library is augmented by amplifying the sequenced genomes using mutagenic PCR.  
     
     
         30 . The method of  claim 28  wherein the complexity of the gene fragment expression library is augmented by expressing the nucleic acid fragments in cells that are modified to mutate nucleic acids.  
     
     
         31 . The method of  claim 24  wherein the complexity of the gene fragment expression library is augmented by expressing the nucleic acid fragment in each of its different reading frames.  
     
     
         32 . The method of  claim 31  wherein the nucleic acid fragments are expressed in a reverse reading frame.  
     
     
         33 . The method of  claim 24  wherein the gene fragment library is a phage display library and wherein said phage display library is assayed by screening the library for peptides that are reactive with particular antibodies by affinity chromatography.  
     
     
         34 . The method of  claim 24  wherein assaying the expression library comprises: 
 (i) expressing a reporter molecule operably under the control of the biological activity in a cell wherein at least a molecule associated with the biological activity comprises polypeptide encoded by a nucleic acid that is placed operably in connection with a promoter;    (ii) incubating the cell from step (i) in the presence of a peptide from the gene fragment expression library under conditions promoting interaction between the peptide and a nucleic acid or a polypeptide associated with the biological activity;    (iii) identifying a peptide that in the presence of the cells is capable of modifying expression of said reporter molecule; and    (iv) selecting the peptide in step (iii) that is not generally recognised as being able to modify expression of said reporter molecule in nature.    
     
     
         35 . The method of  claim 24  wherein the biological activity is antibacterial activity and the gene fragment expression library is transformed or transfected into a first bacterial population of cells and wherein assaying the expression library comprises: 
 (i) growing said first bacterial population for a time and under conditions sufficient for expression of the peptides encoded the gene fragment expression library to occur and for release of the peptides from their cognate fusions;    (ii) contacting the expressed peptides with pathogenic bacteria;    (iii) identifying those sequence(s) that are capable of inhibiting the growth of the pathogenic bacteria, or killing the pathogenic bacteria; and    (iv) selecting those peptides from the identification step in step (iv) that are not associated with the inhibition of growth of the pathogenic bacteria, or killing the pathogenic bacteria in their native environment.    
     
     
         36 . The method of  claim 24  wherein the biological activity is antigenic activity or immunogenic activity.  
     
     
         37 . The method of  claim 36  wherein the biological activity is the binding of immunoglobulin E to mast cells.  
     
     
         38 . The method of  claim 24  wherein the peptide is a modulator or mediator of a biological activity.  
     
     
         39 . The method of  claim 38  wherein the peptide is an antagonist of a biological activity.  
     
     
         40 . The method of  claim 24  wherein the peptide that has the biological activity is further assayed by: 
 (i) aligning the sequences of the amino acids of any peptides identified in the method of any of the preceding claims; and    (ii) identifying consensus motifs.    
     
     
         41 . The method of  claim 24  further comprising the step of isolating the identified peptide.  
     
     
         42 . The method of  claim 24  further comprising the step of isolating the nucleic acid fragment that encodes the identified peptide.  
     
     
         43 . The method of  claim 24  wherein the gene fragment expression library is produced by a process comprising cloning nucleic acid fragments capable of encoding a set of peptides placed operably under the control of a promoter sequence which is capable of directing the expression of said nucleic acid fragments in a cell, virus particle or bacteriophage particle.