Glutathione beads and GST fusion proteins
View Patent ↗The present invention relates generally to glutathione derivatized beads which are adapted for use in conjunction with glutathione-S-transferase fusion proteins (generally, GST fusion proteins, which contain a fluorescent label such as fluorescent green protein) for use in flow cytometry. The present invention also relates to methods for detecting and/or quantifying interactions between a GST fusion protein and their binding partners, in particular, labeled binding partners such as fluorescently labeled binding partners. By creating glutathione beads with an appropriate high or increased site density, disadvantages often associated with low affinity systems and quick off-rates in solution may be resolved to provide a workable system and method. Methods of identifying potential agonists, antagonists and regulator compounds of proteins fused to GST from libraries of compounds represents another aspect of the present invention.
1. A high site density glutathione derivatized flow cytometer bead comprising glutathione molecules covalently linked through a crosslinking agent to a polymeric bead, said bead having a diameter ranging from about 1 microns to about 30 microns, said bead having a high site binding density.
2. The derivatized flow cytometer bead according to claim 1 wherein said binding site density of said bead ranges from about 7.5×10 3 binding sites/μm 2 to about 5×10 5 binding sites/μm 2 .
3. The derivatized flow cytometer bead according to claim 1 wherein said binding site density of said bead ranges from about 1×10 4 binding sites/μm 2 to about 1×10 5 binding sites/μm 2 .
4. The derivatized flow cytomer bead according to claim 1 wherein said binding site density of said bead ranges from about 1×10 4 binding sites/μm 2 to about 5×10 4 binding sites/μm 2 .
5. The derivatized flow cytometer bead according to claim 1 wherein said crosslinking agent is linked to said polymeric bead through a hydroxyl group or an amine group on said polymer bead.
6. The derivatized flow cytometer bead according to claim 1 wherein said crosslinking agent has two electrophilic groups or an electrophilic group and a nucleophilic group.
7. The derivatized flow cytometer bead according to claim 6 wherein said crosslinking agent has two electrophilic groups.
8. The derivatized flow cytometer bead according to claim 1 wherein said crosslinking agent is a bis-epoxide crosslinking agent.
9. The derivatived flow cytometer bead according to claim 7 wherein said crosslinking agent is 1,4-butanediyl diglyciyl ether.
10. The derivatized flow cytometer bead according to claim 7 wherein said crosslinking agent is succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC).
11. The derivatized flow cytometer bead according to claim 7 wherein said crosslinking agent is SMCC.
12. The derivatized flow cytometer bead having a diameter of about 3 microns to about 10 microns.
13. The derivatized flow cytometer bead wherein said polymeric bead comprises a polymeric material selected from the group consisting of amino-polystyrene, latex, polycarbohydrate and glass.
14. The derivatized flow cytometer bead according to claim 1 wherein said polymeric bead comprises a crosslinked agarose-dextran composite (Superdex).
15. The derivatized flow cytometer bead according to claim 13 wherein said polymeric material is amino-polystyrene.
16. The derivatized flow cytometer bead according to claim 1 further comprising a GST-fusion protein bound thereto.
17. The derivatized flow cytometer bead according to claim 16 wherein said GST fusion protein comprises GST and a fused protein.
18. The derivatized flow cytometer bead according to claim 17 wherein said fused protein is Bcl-2 protein.
19. The derivatized flow cytometer bead according to claim 17 wherein said fused protein is bound to a binding partner of said protein.
20. The derivatized flow cytometer bead according to claim 17 wherein said binding partner is a protein, peptide, DNA, RNA or a small molecule.
21. The derivatized flow cytometer bead according to claim 17 wherein said fused protein is a Bcl-2 protein.
22. The derivatized flow cytometer bead according to claim 21 wherein said Bcl-2 protein is Bcl-2, Bcl-XL, Bfl-1, Mcl-1, Bcl-W or Bcl-B.
23. The derivatized flow cytometer bead according to claim 19 wherein said fused protein is a Bcl-2 protein.
24. The derivatized flow cytometer bead according to claim 23 wherein said fused protein is Bcl-2, Bcl-XL, Bfl-1, Mcl-1, Bcl-W or Bcl-B.
25. The derivatized flow cytometer bead according to claim 19 wherein said binding partner is fluorescently labeled.
26. The derivatized flow cytometer bead according to claim 25 wherein said binding partner is a fluorescently labeled peptide.
27. The derivatized flow cytometer bead according to claim 26 wherein said peptide is a BH3 peptide based upon sequences found in Bcl-2 proteins.
28. The derivatized flow cytometer bead according to claim 27 wherein said peptide is a Bim BH3 peptide.
29. The derivatized flow cytometer bead according to claim 27 wherein said peptide is a Bid BH3 peptide.
30. The derivatized flow cytometer bead according to claim 27 wherein said peptide is a PUMA BH3 peptide.
31. The derivatized flow cytometer bead according to claim 27 wherein said peptide is a Bak BH3 peptide.
32. The derivatized flow cytometer bead according to claim 19 where said fused protein is a Bcl-2 protein and said binding partner is FITC conjugated epigallocachechin (ECGC).
33. The derivatized flow cytometer bead according to claim 19 wherein said fused protein is a GTPase.
34. The derivatized flow cytometer bead according to claim 33 wherein said GTPase is a Rab, Rac, Rho, Cdc42 or Ras GTPase.
35. The derivatized flow cytometer bead according to claim 34 wherein said binding partner is GTP.
36. The derivatized flow cytometer bead according to claim 34 wherein said GTPase is a Rab GTPase.
37. The derivatized flow cytometer bead according to claim 36 wherein said binding partner is HVps150, hVps34 or a myotubularin.