Expression of soluble therapeutic proteins
View Patent ↗The present invention provides enhanced methods of producing soluble, active fibroblast growth factor-20 (FGF-20), FGF-21, neurotrophin-3 (NT-3), growth hormone (GH), granulocyte colony stimulating factor (G-CSF), or glucocerebrosidase proteins in microorganisms that have an oxidizing environment.
1. A method of producing soluble fibroblast growth factor 21 (FGF-21) in a microorganism, wherein the microorganism has an oxidizing intracellular environment, which method comprises the steps of
a) expressing in the microorganism a nucleic acid that encodes FGF-21 and
b) growing the microorganism at a temperature of 15-20° C. under conditions that allow production of soluble FGF-21.
2. The method of claim 1 , wherein the microorganism is an E. coli.
3. The method of claim 2 , wherein the E. coli has a mutation in the txrB gene and a gor gene.
4. The method of claim 2 , wherein the nucleic acid is expressed under the control of an inducible promoter.
5. The method of claim 1 , wherein the microorganism has a mutation in an endogeneous reductase nucleic acid.
6. The method of claim 1 , further comprising the step of isolating the soluble FGF-21.
7. The method of claim 1 , wherein the soluble FGF-21 is produced on a commercial scale.
8. The method of claim 1 , wherein the FGF-21 comprises a purification tag.
9. The method of claim 1 , wherein the microorganism comprises a heterologous protein disulfide isomerase (PDI).
10. The method of claim 1 , wherein the microorganism comprises a heterologous chaperone protein.
11. The method of claim 1 , wherein the FGF-21 exhibits enzymatic or biological activity.
12. The method of claim 1 , wherein the oxidizing intracellular environment has a ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) that is less than 40.