Sensors and assays for ubiquitin or ubiquitin-like proteins
View Patent ↗The present invention provides compositions comprising chimeric polypeptides that bind to free ubiquitin proteins or free ubiquitin-like proteins with high affinity, as well as chimeric polypeptides that bind to both free and conjugated ubiquitin proteins or free and conjugated ubiquitin-like proteins, and methods of using the chimeric polypeptides to determine the amount of free or total ubiquitin or free or total ubiquitin-like proteins in various types of samples.
1. A chimeric polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, wherein said chimeric polypeptide binds a ubiquitin protein monomer in non-overlapping regions of said ubiquitin protein monomer.
2. The chimeric polypeptide of claim 1 , further comprising a detectable label attached to said chimeric polypeptide.
3. A chimeric polypeptide comprising:
(a) a first polypeptide that binds a ubiquitin protein monomer and a second polypeptide that binds said ubiquitin protein monomer, wherein said first and second polypeptide bind non-overlapping regions of said ubiquitin protein monomer, and
(b) a first linker, wherein said first linker connects said first and said second polypeptide,
wherein said first polypeptide binds to the ubiquitin hydrophobic patch of said ubiquitin protein monomer and comprises a ubiquitin binding domain selected from the group consisting of: Ubiquitin Associated domain (UBA), Ubiquitin Interacting Motif (UIM), double-sided ubiquitin-interacting motif (DUIM), Motif Interacting with Ubiquitin (MIU), coupling of ubiquitin conjugation to ER degradation (CUE), Golgi-localized, Gamma-ear-containing, Arf-binding (GAT), Jun kinase activation domain binding/Mpr1p and Pad1p N-termini (Jab1/MPN), Np14 zinc finger (NZF), ubiquitin-binding zinc finger (UBZ), Ubiquitin binding surface (UBS), and Ubiquitin binding motif (UBM);
wherein said second polypeptide binds to the surface of said ubiquitin protein monomer near Asp58 and comprises the Ruz domain of Rabex-5.
4. The chimeric polypeptide of claim 3 , wherein said first polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, and said second polypeptide comprises the amino acid sequence of SEQ ID NO: 10.
5. The chimeric polypeptide of claim 4 , further comprising:
(c) a third polypeptide that binds to said ubiquitin protein monomer, wherein said first, second and third polypeptides bind to non-overlapping regions of said ubiquitin protein monomer; and
(d) a second linker, wherein said second linker connects said third and said first polypeptide,
wherein said third polypeptide comprises the zinc finger binding domain of Isopeptidase T.
6. The chimeric polypeptide of claim 5 , wherein said third polypeptide comprises an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: 31, amino acid residues 1-147 of the amino acid sequence of SEQ ID NO: 1 and amino acid residues 1-148 of the amino acid sequence of SEQ ID NO: 2.
7. The chimeric polypeptide of claim 3 , further comprising a detectable label attached to said chimeric polypeptide.
8. The chimeric polypeptide of claim 4 , further comprising a detectable label attached to said chimeric polypeptide.
9. The chimeric polypeptide of claim 5 , further comprising a detectable label attached to said chimeric polypeptide.
10. The chimeric polypeptide of claim 6 , further comprising a detectable label attached to said chimeric polypeptide.