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 . A gene fragment expression library comprising a plurality of different nucleotide sequences from a plurality of biodiverse organisms each having a sequenced genome and wherein the organisms are microorganisms and/or eukaryotes having compact genomes. 
     
     
         2 . The library according to  claim 1 , wherein the sequence fragments of the library are from organisms selected from the group consisting of  Fugu rubripes, Caenorhabditis elegans, Saccharomyces cerevisiae, E. coli, Aquifex aelitcus, Methanococcus jannaschii, Bacillus subtilis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningiditis, Synechocystis  sp.  Bordetella pertussis, Pasteurella multocida, Pseudomonas aeruginosa, Borrelia burgdorferi, Methanobacterium thermoautotrophicum, Mycoplasma pneumoniae, Archaeoglobus fulgidis  and  Vibrio harveyi.    
     
     
         3 . The gene fragment expression library according to  claim 1  constructed using adapted fragments of pooled genomic DNA from an evolutionarily diverse panel of compact genomes. 
     
     
         4 . The expression library according to  claim 1  comprising fragments of DNA from a diverse panel of microorganisms. 
     
     
         5 . The expression library according to  claim 1  comprising adapted fragments of pooled genomic DNA from an evolutionarily diverse set of compact genomes. 
     
     
         6 . The expression library according to  claim 2  wherein the nucleic acid fragments of the library comprise 90 base pairs to 120 base pairs in length. 
     
     
         7 . The expression library according to  claim 2  wherein the nucleic acid fragments of the library are of sufficient length to encode peptides comprising about 30 amino acids in length. 
     
     
         8 . A method of producing a gene fragment expression library comprising a plurality of different nucleotide sequences from different organisms said method comprising producing nucleotide sequence fragments from a nucleotide sequence of known nucleotide composition wherein said nucleotide sequence is from a sequenced genome of a microorganism and/or a sequenced compact genome of an eukaryotic species. 
     
     
         9 . The method according to  claim 8  comprising:
 (i) producing defined fragments of DNA from a diverse panel of microorganisms; 
 (ii) pooling the fragments in direct proportion to the size and complexity of the each genome; and 
 (iii) inserting the combined fragments into an expression vector. 
 
     
     
         10 . The method of  claim 9  wherein the nucleic acid fragments are 90 base pairs to 120 base pairs in length. 
     
     
         11 . The method of  claim 9  comprising digesting the pooled fragments with one or more restriction endonucleases to produce nucleic acid fragments of 90 base pairs to 120 base pairs in length. 
     
     
         12 . The method according to  claim 10  wherein the nucleic acid fragments are of sufficient length to encode peptides comprising about 30 amino acids in length. 
     
     
         13 . The method according to  claim 11  wherein the nucleic acid fragments are of sufficient length to encode peptides comprising about 30 amino acids in length. 
     
     
         14 . The method according to  claim 9  comprising:
 (i) producing adapted fragments of pooled genomic DNA from an evolutionarily diverse set of compact genomes wherein the relative concentration of DNA in the pool from larger genomes is increased in proportion to the total haploid genome size; and 
 (ii) inserting the combined fragments into an expression vector. 
 
     
     
         15 . The method according to  claim 14  comprising fragmenting the pooled genomic DNA by mechanical shearing. 
     
     
         16 . The method of  claim 14  comprising producing the pooled genomic DNA by polymerase extension of partially degenerate oligonucleotides annealed to denatured genomic DNA and amplification using polymerase chain reaction (PCR). 
     
     
         17 . The method of  claim 16  wherein PCR is mutagenic PCR.