Methods of constructing and screening diverse expression libraries
The present invention provides novel methods for producing nucleic acid fragment libraries that express highly diverse peptides or protein domains and, in particular, methods for producing nucleic acid fragment libraries wherein the nucleic acid fragments of the libraries are derived from two or more diverse characterized genomes.
1. A method of constructing an expression library for expressing a peptide having a conformation sufficient for binding to a target protein or nucleic acid, said method comprising:
(a) producing nucleic acid fragments from nucleic acids derived from two or more microorganisms or eukaryotes containing compact genomes, each of said microorganisms or eukaryotes having a substantially sequenced genome; and
(b) selecting nucleic acid fragments of sufficient length to encode a peptide comprising a protein domain having an average length of at least 12 to 15 amino acids; and
(c) inserting the nucleic acid fragments at (b) into a suitable expression construct in an amount proportional to the size of the genome from which the fragments were derived thereby producing recombinant constructs, wherein each fragment is in operable connection with a promoter sequence that is capable of conferring expression of that fragment.
2. The method of claim 1 further comprising selecting nucleic acid fragments from the fragments at (a) that have substantially different nucleotide sequences thereby enhancing nucleotide sequence diversity among the selected fragments compared to the diversity of sequences in the genome.
3. The method of claim 1 , further comprising introducing the recombinant construct into a host cell.
4. The method according to claim 1 wherein producing nucleic acid fragments comprising amplifying nucleic acid from two or more microorganisms or eukaryotes comprising compact genomes.
5. The method according to claim 1 wherein producing nucleic acid fragments comprises amplifying genomic DNA from two or more microorganisms or eukaryotes comprising compact genomes.
6. The method according to claim 1 wherein step 1(c) comprises inserting the nucleic acid fragments at (b) into a suitable expression construct in equimolar amounts thereby producing recombinant constructs, wherein each fragments is in operable connection with a promoter sequence that is capable of conferring expression on that fragment.
7. The method according to claim 1 wherein the two or more microorganisms or compact eukaryotes are selected from the group consisting of Aeropyrum pernix, Anopheles gambiae, Arabidopsis thaliana, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Caenorhabditis elegans, Chlamydia trachomatis, Danio rerio, Drosophila melanogaster, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methemobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyococcus horikoshii, Saccharomyces cerevesiae, Schizosaccharomyces pombe, Synechocystis PCC 6803 , Takifugu rubripes, Thermoplasma volcanium, and Thermotoga maritima.
8. The method according to claim 1 wherein the two or more microorganisms are selected from the group consisting of Archaeoglobus fulgidis, Aquifex aeolicus, Aeropyrum pernix, Bacillus subtilis, Bordetella pertussis TOX6, Borrelia burgdorferi, Chlamydia trachomatis, Escherichia coli K12, Haemophilus influenzae (rd), Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningtidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Synechocystis PCC 6803 , Theroplasma volcanium and Thermotoga maritima.
9. The method according to claim 1 wherein the promoter sequence is operable in a yeast cell.
10. The method according to claim 1 wherein the promoter sequence is operable in a bacterial cell.
11. The method according to claim 1 wherein the expression construct is a yeast expression vector.
12. The method according to claim 1 wherein the expression construct is a phage display vector.
13. The method according to claim 3 wherein the host cell is a yeast cell.
14. The method according to claim 3 wherein the host cell is a bacterial cell.