Fibrinogen binding moieties
Compositions comprising non-naturally occurring fibrinogen binding moieties are described, together with methods of use thereof, e.g., for detecting or isolating fibrinogen molecules in a solution, for blood circulation imaging, and for linking therapeutics or other molecules to fibrinogen. Preferred binding peptides having a high affinity for fibrinogen are particularly disclosed.
1. A fibrinogen binding moiety comprising a polypeptide comprising an amino acid sequence of the formula:
wherein: Xaa 2 is Asn, Met, or Ser; Xaa 3 is Ala or Ser; Xaa 4 is Arg, Asn, or Tyr; Xaa 5 is Pro, Thr, or Trp; Xaa 6 is Ile, Met, or Tyr; Xaa 7 is Ala, His, or Ser; Xaa 8 is Leu, Pro, or Tyr; and Xaa 9 is Trp or Tyr.
2. The binding moiety according to claim 1 , wherein said polypeptide comprises an amino acid sequence selected from the group consisting of:
3. The binding moiety according to claim 1 , wherein said polypeptide comprises an amino acid sequence of the formula:
Ac-AEGTGSLYLCNSYPMHPYCNPGK-NH 2 (SEQ ID NO:48)
where Ac— denotes acetylation and —NH 2 denotes amidation.
4. A fibrinogen binding moiety according to any one of claims 1 or 3 , wherein said polypeptide is expressed by a recombinant bacteriophage.
5. A composition of matter comprising a binding moiety according to any one of claims 1 or 3 linked to a solid support material selected from the group consisting of cellulose, plastic, metal, rubber, wood, nylon, glass, acrylamide, agarose, and combinations thereof.
6. The composition according to claim 4 , wherein said binding moiety is linked to a chromatographic matrix material.
7. A composition, of matter comprising a binding moiety according to any one of claims 1 or 3 linked to a detectable label.
8. The composition according to claim 7 , wherein said detectable label is selected from the group consisting of radionuclides, detectable proteins, epitope tags, biotin, streptavidin, enzymes, antibodies, and fluorescent labels.
9. The composition according to claim 8 , wherein the detectable label is a technetium-containing compound.
10. A composition of matter comprising a binding moiety according to any one of claims 1 or 3 linked to a drug, biopharmaceutical, or polypeptide of interest.
11. A method for detecting fibrinogen in a solution comprising: (a) contacting said solution with a binding moiety according to any one of claims 1 or 3 under conditions wherein said binding moiety will form a complex with said fibrinogen, and (b) detecting said complex.
12. The method according to claim 11 , wherein the solution is blood and the binding moiety is linked to a detectable label.
13. A method for isolating fibrinogen from a solution containing it comprising: (a) immobilizing a composition according to any one of claims 1 or 3 on a solid support; (b) contacting a solution containing fibrinogen with said solid support of (a) under conditions wherein said binding moiety will form a complex with said fibrinogen; and, thereafter, (c) separating the solid support from the unbound components of said solution.
14. The method according to claim 13 , wherein said solid support is selected from the group consisting of chromatographic matrix materials, filters, magnetic beads, and the surface of a plastic or glass container.
15. The method according to claim 13 , further comprising the step: (d) eluting and recovering the fibrinogen from said support.
16. The method according to claim 15 , wherein the eluant used in step (d) is a citrate buffer or a glycine buffer.
17. The method according to claim 13 , wherein the solution is selected from the group consisting of a recombinant eukaryotic or prokaryotic cell extract, and whole blood.
18. A method for isolating fibrinogen from a solution containing it comprising: (a) contacting a solution containing fibrinogen with a binding moiety according to any one of claims 1 or 3 , in which said binding moiety is conjugated to an affinity ligand, under conditions suitable for formation of a binding complex between said binding moiety and said fibrinogen; (b) contacting the solution of step (a) with an immobilized binding partner for said affinity ligand under conditions suitable for formation of a binding complex between the affinity ligand and the binding partner; and (c) removing unbound materials in the solution from the complex formed in step (b).
19. The method according to claim 18 , wherein said affinity ligand is a polyhistidine tag.
20. The method according to claim 18 , wherein said affinity ligand is biotin and said immobilized binding partner for said affinity ligand is streptavidin.
21. A method for assessing blood circulation in a subject comprising: (a) introducing into the circulatory system of the subject a composition comprising a detectable binding moiety according to claim 7 , and (b) detecting any circulation of complexes formed between said detectable binding moiety and fibrinogen in the circulatory system of said subject.
22. The method according to claim 21 , wherein said detectable compound consists essentially of a complex of said detectably labeled binding moiety and fibrinogen.
23. The method according to claim 21 , wherein the binding moiety is detectably labeled with a magnetic resonance imaging agent.