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US 12,203,103 B2Granted
Selection marker for cell transfection and protein production
AssigneeSanofi
InventorsBruno Dumas, Nabil Lounis
Life of this patent
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Abstract
The present invention is within the field of industrial protein production. The invention provides a novel expression system using dehydroorotate dehydrogenase (DHODH) as a selection marker in combination with leflunomide or a metabolite thereof, notably for use in mammalian cell lines. Expression vectors encoding DHODH, cell lines comprising said vectors and methods of producing recombinant proteins are also provided.
Claims7
A method of isolating a clone cell that produces a recombinant protein, the method comprising:
- (a) transfecting a cell line with an expression vector comprising a nucleotide sequence encoding a mammalian dihydroorotate dehydrogenase (DHODH) and at least one expression cassette for expressing a recombinant protein, wherein the nucleotide sequence is at least 90% identical to the nucleotide sequence of SEQ ID NO:
- 1 or the nucleotide sequence of SEQ ID NO:
- 3;
- (b) culturing the transfected cell line under conditions suitable for production of the recombinant protein;
- and
- (c) isolating a clone cell that produces the recombinant protein;
- wherein the DHODH is inhibited by at least one DHODH inhibitor;
- wherein the transfected cell line is cultured in the presence of at least one DHODH inhibitor, wherein the concentration of the at least one DHODH inhibitor is increased during step (b);
- wherein the at least one DHODH inhibitor is terflunomide;
- and
- wherein the concentration of teriflunomide is between 25 μM and 200 μM.
8 more elements
Dependencies are inferred from the claim text.