High pressure refolding of protein aggregates and inclusion bodies
View Patent ↗The present disclosure provides an effective method for the refolding of denatured proteins in solution so that properly folded, biologically active protein in solution is recovered in high yield. The refolding takes place at pressures between about 0.25 kbar to about 3.5 kbar, advantageously at about 1.5 kbar to about 3 kbar. Typically a chaotropic agent is present at a concentration which is not effective for denaturing protein at atmospheric pressure, and optionally, oxidation-reduction reagents can be incorporated in the refolding solution so that native intramolecular disulfide bonds can be formed where that is desired. The method is applicable to substantially all proteins, especially after solubilization and/or denaturation of insoluble protein aggregates, inclusion bodies, or abnormal oligomeric (soluble) aggregates.
1. A method for producing disaggregated biologically active protein from a mixture comprising aggregated protein comprising the steps of:
(a) adjusting total protein concentration in the aggregate mixture to from about 0.01 mg/mL to about 500 mg/mL; then
(b) increasing the pressure on the aggregate mixture to from about 0.25 kbar to about 12 kbar for a time and temperature sufficient for disaggregation of the protein; then
(c) incubating the mixture under pressure in the range from about 0.25 kbar to about 3.3 kbar; then
(d) reducing the pressure to atmospheric pressure, whereby protein in the mixture remains disaggregated and biological activity is retained;
wherein said pressure is reduced at a controlled depressurization rate.
2. The method of claim 1 , wherein said controlled depressurization rate is from about 10 bar/minute to about 13.3 bar/minute.
3. The method of claim 2 , wherein said controlled depressurization rate is about 10 bar/minute.
4. The method of claim 2 , wherein said controlled depressurization rate is about 13.3 bar/minute.
5. The method of claim 1 , wherein during the incubation step (c), the protein concentration is from about 1 to about 100 mg/mL.
6. The method of claim 2 , wherein during the incubation step (c), the protein concentration is from about 1 to about 100 mg/mL.
7. The method of claim 3 , wherein during the incubation step (c), the protein concentration is from about 1 to about 100 mg/mL.
8. The method of claim 4 , wherein during the incubation step (c), the protein concentration is from about 1 to about 100 mg/mL.
9. The method of claim 1 , wherein during the incubation step (c), the protein concentration is from about 1 to about 20 mg/mL.
10. The method of claim 2 , wherein during the incubation step (c), the protein concentration is from about 1 to about 20 mg/mL.
11. The method of claim 3 , wherein during the incubation step (c), the protein concentration is from about 1 to about 20 mg/mL.
12. The method of claim 4 , wherein during the incubation step (c), the protein concentration is from about 1 to about 20 mg/mL.
13. The method of claim 1 , wherein, prior to step (a), the aggregated protein is treated with a reducing agent.
14. The method of claim 1 , further comprising adding, prior to step (b), a chaotropic agent at a concentration up to about 8 M.
15. The method of claim 1 , further comprising adding, prior to step (b), a chaotropic agent at a concentration up to about 1.0 M.
16. The method of claim 15 , wherein the chaotropic agent is guanidinium hydrochloride.
17. The method of claim 1 , wherein after step (c), the concentration of the chaotropic agent is decreased to less than about 0.1 M.