Artificial binding proteins based on ubiquitin
The invention relates to modified proteins of the superfamily of “ubiquitin-like proteins”, proteins that have a ubiquitin-like fold and fragments or fusion proteins thereof. As a result of said modification, the proteins have a binding affinity with respect to a predetermined binding partner that did not exist previously. The invention also relates to a method for the production and utilization of said proteins.
1. A modified ubiquitin protein comprising a ubiquitin-like folding motif, wherein:
the modified ubiquitin protein comprises substitutions of four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 of a ubiquitin protein having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15,
wherein
the modified ubiquitin protein binds to a predetermined binding partner, wherein the predetermined binding partner exhibits a binding affinity with respect to the modified ubiquitin that did not exist previously with respect to a ubiquitin polypeptide of SEQ ID NO: 35, and wherein
the modified ubiquitin protein has a dissociation constant (K D ) of from 10 −5 M to 10 −12 M with respect to the binding partner.
2. The modified ubiquitin protein of claim 1 , wherein Gln62, Lys63, Glu64, Ser65 and Thr66 in the protein having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15 are modified.
3. The modified ubiquitin protein of claim 1 , wherein the four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 that are modified include Gln62, Lys63 or both Gln62 and Lys63.
4. The modified ubiquitin protein of claim 1 , wherein the protein having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15 is human ubiquitin or another mammalian ubiquitin, and further wherein four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 are modified so that these modified amino acids comprise the region with binding affinity to the binding partner.
5. The modified ubiquitin protein of claim 4 , wherein Gln2, Phe4, Lys6, Glu64, Ser65 and Thr66 are modified.
6. The modified ubiquitin protein of claim 4 , wherein Gln62, Lys63, Glu64, Ser65 and Thr66 are modified.
7. The modified ubiquitin protein of claim 4 , wherein the four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 that are modified include Gln62, Lys63 or both Gln62 and Lys63.
8. The modified ubiquitin protein of claim 1 , wherein the amino acids are modified by random mutagenesis.
9. The modified ubiquitin protein of claim 1 , wherein the binding partner of interest is an antigen or a hapten.
10. The modified ubiquitin protein of claim 1 , wherein the binding affinity, expressed in K D , to the binding partner of interest is 10 −6 M to 10 −12 M, 10 −8 to 10 −12 M, or 10 −9 to 10 −12 M.
11. The modified ubiquitin protein of claim 1 , wherein the modified ubiquitin protein is produced by a method comprising:
(a) selecting a protein to be modified having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15;
(b) providing a binding partner of interest, wherein the binding partner of interest exhibits a binding affinity with respect to the modified ubitquitin that did not exist previously with respect to the protein having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15;
(c) selecting four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 of the protein to be modified having the amino acid sequence comprising SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15;
(d) modifying four to eight amino acids at positions Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 and Thr66 by substitution, and further wherein the ubiquitin-like folding motif present in the protein to be modified having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15 is maintained in the modified ubiquitin protein;
(e) contacting the modified protein with the binding partner of interest provided in step (b); and
(f) detecting a modified protein having a binding affinity with the binding partner predetermined in step (b).
12. The modified ubiquitin protein of claim 11 , wherein the method of producing the modified protein further comprises producing a complex comprising bivalent or bispecific binding properties, respectively, by linking the modified ubiquitin protein in a site-specific and covalent manner to a second protein of the same or a different specificity.
13. The modified ubiquitin protein of claim 1 , wherein the amino acid substitutions comprise four or more of Gln2Phe, Phe4Trp, Lys6Val, Gln62Gly, Lys63His, Glu64Gln, Ser65Arg, and Thr66Gly.
14. The modified ubiquitin protein of claim 1 , further comprising an additional substitution or deletion at Gly75 or Gly76 of the protein having the amino acid sequence of SEQ ID NO: 35 or the amino acid sequence that is encoded by SEQ ID NO: 15.
15. The modified ubiquitin protein of claim 1 , wherein the protein of SEQ ID NO: 35 or that is encoded by SEQ ID NO: 15 is a mammalian ubiquitin and the amino acids that are modified amino are acids Gln62, Lys63, Glu64 and Ser65 of the mammalian ubiquitin of SEQ ID NO: 35 or that is encoded by SEQ ID NO: 15.
16. The modified ubiquitin protein of claim 1 , wherein at least two amino acids selected from Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 or Thr66 that are adjacent to each other in the structure are modified.
17. The modified ubiquitin protein of claim 1 , wherein at least two amino acids selected from Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 or Thr66 not adjacent to each other in the structure are modified.
18. The modified ubiquitin protein of claim 1 , wherein three amino acids selected from Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 or Thr66 that are directly adjacent to each other in the sequence are modified.
19. The modified ubiquitin protein of claim 1 , wherein four amino acids selected from Gln2, Phe4, Lys6, Gln62, Lys63, Glu64, Ser65 or Thr66 that are directly adjacent to each other in the sequence are modified.
20. The modified ubiquitin protein of claim 1 , wherein the modified protein is present in a library.
21. The modified ubiquitin protein of claim 20 , wherein the library is an expression library.
22. The modified ubiquitin protein of claim 20 , wherein each of the plurality of modified proteins comprises substitutions in at least four (4) of amino acids 2, 4, 6, and 62-66 of SEQ ID NO: 35.