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1. (WO1993019084) REPLIEMENT ET PURIFICATION DE FACTEURS DE CROISSANCE I RESSEMBLANT A L'INSULINE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

We claim:
1. A method for producing a biologically active recombinant IGF-I, comprising the steps of:
a) obtaining a solution of recombinant IGF-I expressed by a prokaryotic cell;
b) adding a first reducing agent to the solution to form a reduced solution;
c) adding a denaturing agent simultaneously with or after the first reducing agent to form a denatured solution;
d) adding an oxidizing agent to the denatured solution to form an oxidized solution;
e) adding a second reducing agent to the oxidized solution to produce the biologically active recombinant IGF-I.

2. The method of claim 1, wherein the prokaryotic cell is a bacterium.

3. The method of claim 2, wherein the bacterium is E_-_ coli.

4. The method of claim 3, wherein the E^ coli contains 010 (TC3 ) mutIGF-IpT5T.

5. The method of claim 1, wherein the oxidizing agent is a disulfide containing compound.

6. The method of claim 5, wherein the disulfide containing compound is oxidized glutathione or cystine.

7. The method of claim 1, wherein the reducing agent of step (b) is selected from the group consisting of dithiothreitol (DTT) , 2-mercaptoethanol, and dithioerythritol.

8. The method of claim 1, wherein the denaturing agent is guanidine or urea.

9. The method of claim 1, wherein the second reducing agent of step (e) is a thiol-containing reducing reagent.

10. The method of claim 9, wherein the thiol-containing reducing reagent is selected from the group consisting of: dithiothreitol (DTT) , 2-mercaptoethanol, dithioerythritol, cysteine, cystamine, reduced glutathione, and a reducing agent containing an added disulfide containing compound.

11. The method of claim 1, further comprising isolating properly refolded recombinant IGF-I from improperly refolded recombinant IGF-I after step (e) .

12. The method of claim 11, wherein the properly refolded recombinant stimulates proliferation of UMR106 rat osteosarcoma cell line with an ED50 of 2 - 10 ng/ml.

13. The method of claim 1, further comprising:
adding an effective amount of an aminopeptidase after step (e) to cleave the N-terminal methionine; and stopping the reaction.

14. The method of claim 13, wherein the reaction is stopped by lowering the pH of the solution to below pH 5.

15. The method of claim 14, wherein the pH of the solution is lowered to below pH 5 by the addition of trifluoroacetic acid.

16. A pharmaceutical composition comprising biologically active IGF-I prepared according to the method of claim 1 in an acceptable pharmaceutical carrier.

17. A method for treating a patient having an IGF associated condition comprising administering to the patient the biologically active recombinant IGF-I produced in accordance with claim 1.

18. The method of claim 17, wherein the biologically active recombinant IGF-I is in an acceptable pharmaceutical carrier.

19. A method for treating a patient having an IGF associated condition comprising administering to the patient the biologically active recombinant IGF-I produced in accordance with claim 11.

20. The method of claim 19, wherein the biologically active recombinant IGF-I is in an acceptable pharmaceutical carrier.