OB fold domains
Provided herein are modified OB-fold domains having desired properties; methods of producing libraries of modified OB-fold domains; the libraries of modified OB-fold domains produced by such methods; methods for screening such libraries of modified OB-fold domains for desired biological activities; and the modified OB-fold domains identified from such libraries. Provided herein are modified OB-fold domains obtainable from Pyrobaculum aerophilum that exhibit modified binding interactions.
1. An isolated modified OB-fold domain, obtainable from a naturally occurring OB-fold domain and removed from the naturally occurring protein within which it is contained, wherein:
(a) at least one amino acid residue located in a β-strand of the OB-fold domain binding face is mutated by substitution as compared to the naturally occurring OB-fold domain amino acid residue, or
(b) at least one amino acid residue located in a β-strand of the OB-fold domain binding face and at least one amino acid residue located in a strand of the OB-fold domain loop region are mutated by substitution as compared to the naturally occurring OB-fold domain amino acid residues,
(c) at least one modified amino acid residue in a strand of the OB-fold domain loop region is mutated by substitution as compared to the naturally occurring OB-fold domain amino acid residue,
wherein said isolated modified OB-fold domain is selected from the group consisting of Pyrobaculum aerophilum Asp tRNA synthase OB-fold domain having between 4 and 17 mutated amino acids and Pyrobaculum aerophilum initiation factor IF-5A OB-fold domain having between 2 and 11 mutated amino acid residues;
and wherein said modified OB-fold domain is soluble and has altered binding characteristics as compared to the naturally occurring OB-fold domain, and wherein said domain specifically binds to a different binding partner than the naturally occurring OB-fold domain.
2. The modified OB-fold domain of claim 1 wherein said domain has modified binding with its naturally occurring binding partner.
3. The modified OB-fold domain of claim 1 wherein said mutated amino acid residues are in a β-strand of the fold-related binding face.
4. A method of obtaining a modified OB-fold domain of claim 1 , comprising:
a) obtaining nucleic acid encoding a naturally occurring OB-fold domain, or encoding a portion thereof comprising a strand of the binding face and/or a strand of the loop, and
b) altering the nucleic acid such that it encodes at least one mutated amino acid residue on a β-strand of the binding face and/or at least one mutated amino acid residue on a strand of a loop as compared to the naturally occurring OB-fold domain, wherein a modified OB-fold domain is obtained and wherein said modified OB-fold domain has altered binding as compared to said naturally occurring OB-fold domain.
5. The method of claim 4 further comprising altering nucleic acid encoding said modified OB-fold domain, and/or altering nucleic acid encoding at least one amino acid of a protein that comprises said modified OB-fold domain.
6. A method of producing a library of modified OB-fold domains for display comprising,
a) obtaining nucleic acid encoding an OB-fold domain, or a portion thereof,
b) subjecting said nucleic acid to random alterations, to produce a collection of altered nucleic acid encoding modified OB-fold domains having at least four randomized amino acid residues, and
c) selecting soluble modified OB-fold domains for the library;
wherein said library comprises OB-fold domains according to claim 1 .
7. The method of claim 6 wherein said nucleic acid encodes at least one mutated amino acid residue of a strand of the OB-fold domain binding face and/or a strand of an OB-fold domain loop.
8. The method of claim 6 , further comprising placing the library of altered nucleic acid encoding modified OB-fold domains into a population of host cells capable of displaying said modified OB-fold domains on their surface.
9. An isolated nucleic acid encoding the modified OB-fold domain of claim 1 .
10. A host cell comprising a nucleic acid encoding the modified OB-fold domain of claim 1 .
11. A phage comprising a nucleic acid encoding the modified OB-fold domain of claim 1 .
12. A composition comprising a nucleic acid encoding the modified OB-fold domain of claim 1 .
13. A method of screening a library of modified OB-fold domains according to claim 1 for binding with a binding partner, comprising:
a) obtaining a population of host cells or viral particles displaying a library of modified OB-fold domains according to claim 1 on their surface;
b) contacting said population of host cells or viral particles with said binding partner under conditions suitable for binding of said binding partner to said modified OB-fold domain; and
c) determining binding of said binding partner to said modified OB-fold domain.
14. The method of claim 13 wherein said host cells or viral particles are phage that display the modified OB-fold domains on their surface.
15. A phage library of modified OB-fold domains of claim 1 , wherein said modified OB-fold domains are obtainable from Pyrobaculum aerophilum.
16. A modified OB-fold domain of claim 1 displayed on the surface of a cell or viral particle.
17. The modified OB-fold domain of claim 16 wherein said cell or viral particle is a phage, bacteria or yeast.
18. A modified OB-fold domain of claim 1 attached to a solid support.
19. The modified OB-fold domain of claim 18 wherein said support is selected from the group consisting of beads, glass, slides, chips, and gelatin.