Methods and compositions for enhancing polypeptide production
Methods of increasing production, stability or activity of a target polypeptide are provided herein. The method includes increasing the level of an antioxidant in a cell comprising a polynucleotide encoding the target polypeptide. Also provided are cells and transgenic organisms produced using the methods described herein. Methods of treating a subject with a condition treatable by administration of the target polypeptide are also disclosed. Finally methods and compositions for transiently increasing antioxidants in plant cells are provided.
1. A method of increasing production of a target polypeptide in a plant cell, the method comprising:
co-introducing Vitamin C and a first polynucleotide encoding the target polypeptide into the cell using a transient infiltration or delivery system selected from the group consisting of vacuum infiltration, liposome mediated delivery, sonication, temperature (heat) shock, particle bombardment, electroporation, reversible membrane permeabilization, and microinjection to produce the cell comprising the first heterologous polynucleotide encoding the target polypeptide, wherein both the Vitamin C and polynucleotide encoding the target polypeptide are co-delivered,
wherein the Vitamin C is added at 20 mM to 60 mM, and
wherein the increased level of Vitamin C in the cell increases the production of the target polypeptide and reduces the formation of multimers of the target polypeptide as compared to the production of the target polypeptide in a control cell without increased Vitamin C.
2. The method of claim 1 , wherein the plant cell is a from a plant of the genus Nicotiana.
3. The method of claim 1 , additionally comprising increasing the level of a lipophilic antioxidant in the cell.
4. The method of claim 1 , wherein the first polynucleotide encoding the target polypeptide is being stably integrated into the genome of the cell.
5. The method of claim 1 , wherein the target polypeptide is transiently expressed by the cell.
6. The method of claim 1 , wherein the target polypeptide is an antibody, an interleukin, a growth factor, an enzyme, a cytokine, or a therapeutic polypeptide.
7. The method of claim 1 , wherein the target polypeptide requires post-translational processing for activity.
8. The method of claim 1 , wherein Vitamin C is added at a concentration of 40 mM to 60 mM.
9. The method of claim 3 , wherein the lipophilic antioxidant is added at a concentration of 10 μM to 100 μM.
10. The method of claim 1 , further comprising harvesting the target polypeptide from the cell.
11. The method of claim 9 , wherein the lipophilic antioxidant is vitamin K or vitamin E.