Methods and compositions for extracting proteins from cells
View Patent ↗A process is disclosed for releasing proteins from cells and/or inactivating viruses. In the process, a host cell containing a protein of interest is contacted with a solution of an effective amount of a detergent.
1. A method of releasing a protein of interest from host cells comprising
a. suspending the host cells in a buffer, and
b. adding a detergent to the suspended cells wherein the detergent is an amphipathic charged amine or an amphipathic charged amine oxide, and wherein the detergent is in a concentrated form of at least 3 times the desired final concentration of the detergent after dilution in the buffer of step a.
2. A method of releasing a protein of interest from host cells comprising
a. suspending the host cells in a buffer, and
b. adding a detergent to the suspended cells, wherein the detergent is selected from the group consisting of: tributylphosphate, dimethyldecylamine, dimethyltridecylamine, dimethylundecylamine, dimethyldidecylamine, dimethyltetradecylamine, dimethylhexadecylamine, dimethyldecylamineoxide, dimethylundecylamineoxide, dimethyldidecylamineoxide, dimethyltetradecylamineoxide and dimethyltridecylamineoxide, and wherein the detergent is in a concentrated form of at least 3 times the desired final concentration of the detergent after dilution in the buffer of step a.
3. The method of claim 1 , wherein the detergent is not dimethyltridecylamine.
4. The method of claim 3 , further comprising the step of adding glycerol to the suspended host cells, and wherein the glycerol is in a concentrated form of at least 3 times the desired final concentration of the glycerol after dilution in the buffer of step 1.
5. The method of claim 4 , wherein the detergent comprises a final concentration of between 0.1 to 10 percent.
6. The method of claim 4 , wherein the glycerol comprises a final concentration of between 0.6 to 15 percent.
7. The method of claim 5 , wherein the glycerol comprises a final concentration of between 0.6 to 6 percent.
8. The method of claim 1 , wherein the detergent added is a pure detergent.
9. The method of claim 1 , wherein the host cells are at least 1 kg in weight.
10. The method of claim 1 , wherein the host cells are Pichia pastoris cells.
11. The method of claim 1 , wherein the buffer has an ionic strength of less than 350 mM and at least 1 mM.
12. The method of claim 1 , further comprising the step of incubating the cell and detergent mixture from 90 minutes to 24 hours.
13. The method of claim 12 , wherein the incubation is from 8 hours to 24 hours.
14. The method of claim 1 , further comprising the step of incubating the cell and detergent mixture at a temperature of between about 3° C. and about 10° C.
15. The method of claim 2 , wherein the detergent is not dimethyltridecylamine.
16. The method of claim 3 , further comprising the step of adding glycerol to the suspended host cells, and wherein the glycerol is in a concentrated form of at least 3 times the desired final concentration of the glycerol after dilution in the buffer of step 1.
17. The method of claim 4 , wherein the detergent comprises a final concentration of between 0.1 to 10 percent.
18. The method of claim 4 , wherein the glycerol comprises a final concentration of between 0.6 to 15 percent.
19. The method of claim 5 , wherein the glycerol comprises a final concentration of between 0.6 to 6 percent.