Inducible eukaryotic expression system

摘要

Compositions and methods for the inducible expression of genes in eukaryotic cells are provided. Expression of a nucleotide sequence of interest encoding a protein of interest is controlled by a regulatory fusion protein that consists of a transcription blocking domain and a ligand-binding domain. When a cognate ligand for the ligand-binding domain is present, transcription of the nucleotide sequence of interest is blocked. Upon removal of the cognate ligand, the nucleotide sequence of interest is transcribed. The method is useful for large scale bioreactor production of a desired protein of interest in eukaryotic cells.

主权项

1. A method for expressing a protein of interest from a gene of interest in eukaryotic cells in a bioreactor, comprising:
(a) seeding a starter culture for a bioreactor with eukaryotic cells in the presence of a concentration of a ligand that stabilizes a regulatory fusion protein (RFP) and inhibits expression of a protein of interest, wherein the eukaryotic cells comprise
i. a promoter operably linked to the gene of interest encoding the protein of interest and controlled by a Tet or Arc operator operably linked and positioned 5′ with respect to the promoter, and,ii. a gene encoding the regulatory fusion protein (RFP), wherein the RFP consists essentially of
(1) a transcription blocking domain comprising (i) a TetR repressor or (ii) an Arc repressor DNA-binding domain; and(2) a ligand-binding domain of an estrogen receptor of SEQ ID NO:8,wherein the RFP inhibits expression of the gene of interest by binding to the Tet or Arc operator and inhibiting transcription in the presence of the ligand, but not in the absence of the ligand;wherein the ligand is capable of binding to the ligand-binding domain of an estrogen receptor; (b) growing the cells in the starter culture to establish a culture, wherein expression of the protein of interest is inhibited; (c) seeding a 10 liter bioreactor with the culture, wherein media employed in the 10 liter bioreactor comprises an inhibitory amount of the ligand; (d) conducting a seed train to seed a 3,000 liter or 10,000 liter bioreactor, wherein media added subsequent to the 10 liter bioreactor does not contain the ligand, and wherein the seed train is conducted so that sufficient media is added to reduce the concentration of the ligand at the 3,000 liter or 10,000 liter stage; and (e) growing the cells of the 3,000 liter or 10,000 liter bioreactor under conditions sufficient to express the protein of interest.