Method for obtaining active pro-NGF and beta-NGF
The invention relates to a method for producing biologically active β-NGF from the proform proNGF. After expressing the proform of the β-NGF in a prokaryotic host cell, the recombinant protein is isolated in the form of insoluble inactive aggregates (inclusion bodies). After the solubilization thereof in a strong denaturing agent and the subsequent conversion thereof into the natural conformation, which is determined by the disulfide bridges present in the natural β-NGF, biologically active β-NGF is obtained by subsequently splitting-off the prosequence.
1. A method for the preparation of biologically active proNGF, comprising:
(a) recombinantly expressing a proNGF coding sequence in a prokaryote to produce a proNGF polypeptide comprising its complete prosequence, wherein the proNGF polypeptide comprising its complete prosequence is present in the prokaryote in inclusion bodies;
(b) transferring the inclusion bodies comprising the proNGF polypeptide comprising its complete prosequence into a solution of a denaturing agent to obtain a denatured and solubilized proNGF polypeptide; and
(c) transferring the denatured and solubilized proNGF polypeptide into a non-denaturing or weakly denaturing renaturation solution with a pH of between 7 and 10, wherein the denatured and solubilized proNGF polypeptide remains soluble in the renaturation solution and assumes a biologically active conformation comprising the same disulfide bonds that are present in native proNGF,
whereby biologically active proNGF is obtained.
2. The method of claim 1 , wherein the renaturation solution contains arginine.
3. The method of claim 2 , wherein the concentration of arginine is 0.2 to 1.5 mol/l.
4. The method of claim 2 , wherein the renaturation solution comprises a thiol component in its reduced and oxidized form.
5. The method of claim 3 , wherein the renaturation solution comprises a thiol component in its reduced and oxidized form.
6. The method of claim 2 , wherein guanidinium hydrochloride or urea is used as the denaturing agent.
7. The method of claim 3 , wherein guanidinium hydrochloride or urea is used as the denaturing agent.
8. The method of claim 1 , wherein the renaturation solution comprises a thiol component in its reduced and oxidized form.
9. The method of claim 8 , wherein guanidinium hydrochloride or urea is used as the denaturing agent.
10. The method of claim 1 , wherein guanidinium hydrochloride or urea is used as the denaturing agent.
11. The method of claim 1 , further comprising isolating the biologically active proNGF.
12. A method for the preparation of biologically active β-NGF polypeptide, comprising:
(a) recombinantly expressing a proNGF coding sequence in a prokaryote to produce a proNGF polypeptide comprising its complete prosequence, wherein the proNGF polypeptide comprising its complete prosequence is present in the prokaryote in inclusion bodies;
(b) transferring the inclusion bodies comprising the proNGF polypeptide into a solution of a denaturing agent to obtain a denatured and solubilized proNGF polypeptide;
(c) transferring the denatured and solubilized proNGF polypeptide into a non-denaturing or weakly denaturing renaturation buffer with a pH of between 7 and 10, wherein the proNGF polypeptide remains soluble in the renaturation buffer and assumes a biologically active conformation with the same disulfide bonds present as in native proNGF; and
(d) cleaving off the prosequence,
whereby a biologically active β-NGF polypeptide is prepared.
13. The method of claim 12 , further comprising isolating the biologically active β-NGF polypeptide.
14. The method of claim 12 , wherein the cleaving step comprises employing a protease with a substrate specificity for cleaving after the amino acid arginine.
15. The method of claim 14 , wherein the protease is trypsin.
16. The method of claim 12 , wherein the denaturing agent comprises guanidinium hydrochloride or urea.
17. The method of claim 12 , further comprising isolating the biologically active β-NGF.