Method for the purification of heat shock proteins
Recombinant purified DnaK—having a ATPase activity without the addition of an other chaperone protein—essentially free of T-cell stimulating impurities.
1. A recombinant purified DnaK preparation comprising purified recombinant DnaK,
the preparation
having a purity of 98% or more;
having an ATPase activity without the addition of any other chaperone protein; and
essentially free of T-cell stimulating impurities.
2. The recombinant purified DnaK preparation of claim 1 having
a) a residual DNA contamination ≦1 ng/mg of protein
b) a residual host cell protein contamination (HCP) <5% by weight
c) an endotoxin contamination <0.5 E.U./μg of protein.
3. The recombinant DnaK preparation of claim 1 wherein the content of peptides is below 1% on a molar basis.
4. The recombinant DnaK preparation of claim 1 , wherein the residual DNA contamination is ≦0.5 ng/mg, and/or the HCP is <0.1% by weight, more preferably <0.0001% by weight.
5. The recombinant DnaK preparation of claim 1 , wherein the DnaK is hydrolyzed.
6. The recombinant DnaK preparation of claim 1 , wherein the endotoxin contamination is ≦0.01 E.U./μg of protein.
7. The recombinant purified DnaK preparation of claim 1 , wherein the DnaK is essentially free of nucleotides, in form of a complex with ADP, in form of a complex with ATP or mixtures thereof.
8. A method for the purification of a recombinant DnaK preparation of claim 1 from a cell lysate comprising the steps of:
a) ion exchange chromatography;
b) hydroxylapatite chromatography; and
c) gelatin chromatography.
9. The method of claim 8 wherein DnaK is from saprophytic bacteria, preferably E. coli.
10. The method of claim 8 wherein the ion exchange chromatography is an anion exchange chromatography.
11. The method of claim 8 wherein the hydroxylapatite chromatography is a hydroxylapatite type II chromatography.
12. The method of claim 8 wherein the gelatin chromatography is conducted on gelatin sepharose.
13. A method for forming a complex between recombinant DnaK and at least one peptide or at least one protein comprising the steps of:
a) combining recombinant DnaK of claim 1 with ATP at molar ratio HSP:ATP of 1:1 to 1:10;
b) adding at least one peptide or at least one protein; and
c) incubating at a temperature of 10° C. to 60° C., preferably 20° C. to 45° C.
14. A mixture of recombinant DnaK according to claim 1 and at least one peptide or at least one protein.
15. A pharmaceutical product comprising a recombinant purified DnaK preparation of claim 1 .
16. The mixture of claim 14 wherein the mixture is in the form of a complex.
17. A pharmaceutical product comprising a mixture of claim 14 .
18. The recombinant purified DnaK preparation of claim 1 wherein the presence of the T-cell stimulating impurities is determined at a concentration of 1 μg of the purified recombinant DnaK per ml of blood.
19. The recombinant purified DnaK preparation of claim 1 wherein the lack of the T-cell stimulating impurities is evaluated through observation of no T-cell proliferation up to 30 μg of the purified recombinant DnaK per ml of blood and no TNF-αproduction up to 10 μg of the purified recombinant DnaK per ml of blood.