Cell lines
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.
1. A process for stabilizing a eukaryotic cell line which expresses PylRS and tRNAPyl, wherein the cell line is capable of incorporating a gene encoding a target protein and wherein said target protein comprises one or more non-natural amino acids encoded by an amber codon, wherein said process comprises culturing said cell line in the presence of a decoy amino acid of:
(a) formula VIIA:
wherein K is CO or SO 2 ;
Q=H, C 1-6 alkyl, aryl, heteroaryl, -OC 1-6 alkyl, -OCH 2 aryl, -OCH 2 heteroaryl, C 2-6 alkenyl or -OC 2-6 alkenyl; or
(b) formula VIIB:
wherein G=H;
a=4 or 5; and
R=C 1-6 alkyl, C 2-6 alkenyl, CH 2 aryl, C 2-6 alkynyl, C 1-6 haloalkyl or C 1-6 azidoalkyl,
wherein the viability of said eukaryotic cell line is enhanced in comparison to the viability of said eukaryotic cell line cultured in the absence of said decoy amino acid.
2. A process according to claim 1 wherein the decoy amino acid is selected from:
3. A process according to claim 1 wherein the expression of tRNAPyl occurs under the control of a repressible promoter.
4. A process according to claim 1 wherein the eukaryotic cell line is a mammalian cell line is selected from CHO, HEK293, PERC6, COS-1, COS-7, HeLa, VERO, mouse hybridoma and mouse myeloma cell lines.
5. A process according to claim 1 wherein the nonsense codon is an amber codon.
6. A process according to claim 1 wherein the compound of formula VII is a compound of formula VIIA:
wherein K is CO or SO2;
Q=—H, C1-6alkyl, aryl, heteroaryl OC1-6alkyl, —OCH2aryl, —OCH2heteroaryl, —C2-6alkenyl or —OC2-6alkenyl.