Receptors method for purification of G protein-linked

• US 5,225,543 A
• Filed: 03/28/1991
• Issued: 07/06/1993
• Est. Priority Date: 03/28/1991
• Status: Expired due to Term

• Current Assignee: American Colloid Company (Minerals Technologies Incorporated)
• Original Assignee: American Colloid Company (Minerals Technologies Incorporated)
• Inventors: Corbett, Martin J., Shieh, Hong-Ming, Eppler, Cecil M., Zysk, John R.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): WALSH, STEPHEN G
• Application Number:
  US07/677,003
• Time in Patent Office: 831 Days
• Field of Search: 530/307, 530/311, 530/350, 530/367, 530/387, 530/412, 530/413, 530/415
• US Class Current: 530/413
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748

Abstract

The receptor:ligand complex is applied to a solid phase substrate which binds the complex by specifically binding the ligand. The receptor is dissociated from the substrate bound receptor:ligand complex with an eluant capable of releasing the receptor from the ligand and leaving the ligand bound to the solid phase substrate. GTP containing eluants are effective. The receptor is recovered from the eluate and optionally subjected to further purification.

Claims

1. A method for purification of a receptor polypeptide comprising:

(a) contacting a ligand capable of specifically binding the receptor with cell membranes containing the receptor to form a receptor;

ligand complex;

(b) solubilizing the receptor;

ligand complex;

(c) contacting the solubilized receptor;

ligand complex with a solid phase substrate which binds the receptor;

ligand complex by specifically binding with the ligand;

(d) dissociating the receptor from the substrate bound receptor;

ligand complex with an eluant capable of releasing the receptor from the receptor;

ligand complex to produce an eluate containing a purified receptor.

2. The method of claim 1 in which the receptor'"'"'s specific G protein remains associated with the receptor:

ligand complex up to step (d), and in which the receptor and G protein are both contained in the eluate and by which elution yields both receptor and dissociated G protein.

3. The method of claim 1 which includes the further step of (e) recovering the receptor from the eluate.

4. The method of claim 3 in which the receptor is further purified by contact with a substrate capable of binding a glycoprotein.

5. The method of claim 1 in which the eluant is capable of separating the G protein from the receptor.

6. The method of claim 1 in which the receptor:

ligand complex is solubilized with a bile salt-like detergent.

7. The method of claim 1 in which the ligand is biotinylated.

8. The method of claim 7 in which the substrate of step (c) contains avidin or streptavidin.

9. The method of claim 1 in which the eluant in step (d) contains a dication chelating agent.

10. The method of claim 9 in which the eluant contains EDTA, EGTA or a combination thereof.

11. The method of claim 5 in which the eluant contains a GTP analog.

12. The method of claim 10 in which the eluant also contains a GTP analog.

13. The method of claim 4 in which the receptor is further purified by lectin affinity chromatography.

14. A method for purification of a G protein-linked somatostatin receptor comprising:

(a) contacting a biotinylated somatostatin analog ligand with cellular material containing the somatostatin receptor to form a receptor;

ligand complex;

(b) solubilizing the receptor;

ligand complex in a bile salt-like detergent;

(c) contacting the solubilized receptor;

ligand complex with a substrate containing avidin or streptavidin; and

(d) contacting the bound receptor;

ligand complex with an eluant containing a dicationic chelating agent, or a GTP analog, or both to release the receptor into an eluate.

16. The method of claim 14 in which the receptor is a pituitary somatostatin receptor.

17. The method of claim 14 in which the receptor:

ligand complex is solubilized with deoxycholate.

18. The method of claim 17 in which the deoxycholate is combined with lysolecithin at a concentration of up to about 0.2%.

19. The method of claim 14 in which the eluant contains EDTA, EGTA and GTP-γ

-S.

20. The method of claim 14 in which the receptor is further purified by binding with wheat germ agglutinin and elution with N-N'"'"'-N"-triacetylchitotriose.

15. The method of claim 15 in which the ligand is bioS28 or bioS14.