Fluorescent fusion polypeptides and methods of use
Embodiments of the present invention provide for the facile generation of a stable recombinant fusion polypeptides with intrinsic fluorescent properties. The recombinant antibodies may be suitable for qualitative and/or quantitative immunofluorescence analysis. Generally, the fluorescent polypeptides include a fluorescent domain comprising a C-terminus and an N-terminus; a first antibody domain covalently linked to the C-terminus; and a second antibody domain covalently linked to the N-terminus.
1. A fluorescent fusion polypeptide comprising:
a fluorescent domain comprising:
a monomeric fluorescent polypeptide comprising
a C-terminus; and
an N-terminus;
a first antibody domain covalently linked to the C-terminus of the fluorescent domain, wherein the first antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H ); and
a second antibody domain covalently linked to the N-terminus of the fluorescent domain, wherein the second antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
wherein the N-terminus of first antibody domain and the C-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å.
2. The polypeptide of claim 1 wherein at least one covalent link comprises a linker comprising no more than 10 amino acids.
3. The polypeptide of claim 1 wherein of the fluorescent domain, the first antibody domain, and/or the second antibody domain comprises an affinity tag.
4. The polypeptide of claim 1 wherein the first antibody domain and the second antibody domain specifically bind to the same target molecule.
5. The polypeptide of claim 1 wherein the first antibody domain and the second antibody domain specifically bind to different target molecules.
6. The polypeptide of claim 1 wherein the first antibody domain comprises a variable light chain (V L ) comprising an N-terminus.
7. The polypeptide of claim 1 wherein the second antibody domain comprises a variable heavy chain (V H ) comprising a C-terminus.
8. The polypeptide of claim 7 wherein the first antibody domain comprises a variable light chain (V L ) comprising an N-terminus.
9. The polypeptide of claim 1 wherein the monomeric fluorescent polypeptide comprises at least a portion of a monomeric fluorescent protein sufficient to emit a fluorescent signal.
10. A composition comprising:
a first fluorescent fusion polypeptide comprising:
A. a fluorescent domain comprising:
a monomeric fluorescent polypeptide comprising
a C-terminus; and
an N-terminus;
a first antibody domain covalently linked to the C-terminus of the fluorescent domain of the first fluorescent fusion polypeptide, wherein the first antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H ); and
a second antibody domain covalently linked to the N-terminus of the fluorescent domain of the first fluorescent fusion polypeptide, wherein the second antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
wherein the N-terminus of first antibody domain and the C-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å; and
B. a second fluorescent fusion polypeptide comprising:
a fluorescent domain comprising:
a monomeric fluorescent polypeptide comprising
a C-terminus; and
an N-terminus;
a first antibody domain covalently linked to the C-terminus terminus of the fluorescent domain of the second fluorescent fusion polypeptide, wherein the first antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H ); and
a second antibody domain covalently linked to the N-terminus terminus of the fluorescent domain of the second fluorescent fusion polypeptide, wherein the second antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
wherein the N-terminus of first antibody domain and the C-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å;
wherein the second fluorescent fusion polypeptide binds to a different target and/or emits a second fluorescent signal.
11. The composition of claim 10 wherein the fluorescent domain of the first fluorescent fusion polypeptide emits a first fluorescent signal and the fluorescent domain of the second fluorescent fusion polypeptide emits a second fluorescent signal.
12. The composition of claim 11 wherein the first fluorescent signal comprises a first emission peak, the second fluorescent signal comprises a second emission peak, and the first emission peak is different than the second emission peak.
13. The composition of claim 10 wherein the first fluorescent polypeptide specifically binds to a different target molecule than the second fluorescent fusion polypeptide.
14. A kit comprising:
A. a first container comprising a first fluorescent fusion polypeptide that comprises:
a fluorescent domain comprising:
a monomeric fluorescent polypeptide or comprising
a C-terminus; and
an N-terminus, and that produces a first fluorescent signal comprising a first emission peak;
a first antibody domain covalently linked to the C-terminus of the fluorescent domain, wherein the first antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
a second antibody domain covalently linked to the N-terminus of the fluorescent domain, wherein the second antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
wherein the N-terminus of first antibody domain and the C-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å, and wherein the first fluorescent fusion polypeptide specifically binds to a first analyte; and
B. a second container comprising a second polypeptide that comprises:
a fluorescent domain comprising:
a monomeric fluorescent polypeptide comprising
a C-terminus; and
an N-terminus, and that produces a second fluorescent signal comprising a second emission peak;
a first antibody domain covalently linked to the C-terminus of the fluorescent domain, wherein the first antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H ); and
a second antibody domain covalently linked to the N-terminus of the fluorescent domain, wherein the second antibody domain comprises a variable light chain (V L ) or a variable heavy chain (V H );
wherein the N-terminus of first antibody domain and the C-terminus of the second antibody domain are separated by a distance of no less than 30 Å and no more than 40 Å, and wherein the second fluorescent fusion polypeptide specifically binds to a second analyte.
15. The composition of claim 10 wherein the first antibody domain of the first fluorescent fusion polypeptide comprises a variable light chain (V L ) and the second antibody domain of the first fluorescent fusion polypeptide comprises a variable heavy chain (V H ).
16. The composition of claim 10 wherein the first antibody domain of the second fluorescent fusion polypeptide comprises a variable light chain (V L ) and the second antibody domain of the second fluorescent fusion polypeptide comprises a variable heavy chain (V H ).
17. The kit of claim 14 wherein the first emission peak and the second emission peak are different.