Cell lines
AssigneeMedImmune, LLC
InventorsKenneth H. Grabstein, Michael Van Brunt, Marcello Marelli, William Brady, Jeffrey C. Johnson
Life of this patent
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Abstract
There is provided inter alia a process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl and which is suitable for incorporation of a gene encoding a target protein containing one or more non-natural amino acids encoded by a nonsense codon which comprises culturing said cell line under conditions in which the adverse effect of tRNAPyl expression on cell viability and/or cell growth is reduced or eliminated.
Claims13
A process for preparing a target protein containing one or more non-natural amino adds encoded by an amber codon which comprises:
- (A) introducing into a eukaryotic cell line which expresses PyiRS and tRNAPyl and is capable of incorporating a gene encoding a target protein comprising one or more non-natural amino acids encoded by an amber codon a gene encoding the target protein such that the target protein is stably expressed in the cell line,
- (B) culturing the eukaryotic cell line in the presence of a decoy amino add which is a substrate for PylRS but which is incapable of being incorporated into an extending protein chain, wherein the decoy amino acid is selected from:
- (a) a compound of formula VIIA:
- wherein K is CO or SO2;
- and
- Q=H, C1-6alkyl, aryl, heteroaryl, OC1-6alkyl, OCH2aryl, OCH2heteroaryl, C2-6alkenyl or OC2-6alkenyl;
- and
- (b) a compound of formula VIIB:
- wherein G=H;
- a=4 or 5;
- and
- R═C1-6alkyl, C2-6alkenyl, —CH2aryl, C2-6alkynyl, C1-6haloalkyl or C1-6azidoalkyl;
- and
- (C) expressing the target protein in the presence of one or more non-natural amino adds and in the absence of the decoy amino add.
Dependencies are inferred from the claim text.