Schloßhauer, JeffreyJeffreySchloßhauerZemella, AnneAnneZemellaKubick, StefanStefanKubickCavak, Niño DennisNiño DennisCavakThoring, LenaLenaThoring2025-05-012025-05-012023-02-08https://publica.fraunhofer.de/handle/publica/487187The present invention relates to a method for producing eukaryotic cell lysates comprising at least one exogenous enzyme, wherein the method comprises at least the following steps:a) providing eukaryotic cells transfected with at least one donor template coding for at least one exogenous enzyme which is selected from the group comprising orthogonal aminoacyl-tRNA synthetases, viral RNA polymerases and kinases;b) cultivating the transfected cells of step a) for a predetermined period of time and subsequently harvesting the cells; andc) disrupting the harvested cells and preparing a cell lysate therefrom.More specifically, the invention relates to a method for producing eukaryotic cell lysates which are capable of cell-free synthesis of a target protein comprising a non-canonical amino acid, wherein the method comprises at least the following steps:a) providing eukaryotic cells transfected with at least one donor template coding for an orthogonal aminoacyl-tRNA synthetase, i.e. an aminoacyl-tRNA synthetase which is specific for a tRNA which is not recognized by endogenous aminoacyl-tRNA synthetasen in said eukaryotic cells and is specific for a corresponding non-canonical amino acid;b) cultivating the transfected cells of step a) for a predetermined period of time and subsequently harvesting the cells; andc) disrupting the harvested cells and preparing a cell lysate therefrom.Further aspects of the present invention relate to eukaryotic cell lysates obtainable by the claimed method as well as to novel transfected eukaryotic cells or cell lines expressing at least one orthogonal aminoacyl-tRNA synthetase which may be advantageously used in said method.enpatentEP4130254 A1EP20210189381