Eukaryotic cell lysates comprising exogenous enzymes and methods for preparing the same

Abstract

The present invention relates to a method for producing eukaryotic cell lysates for cell-free protein synthesis 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 and viral RNA polymerases: b) cultivating the transfected cells of step a) for a predetermined period of time and subsequently harvesting the cells; and c) disrupting the harvested cells and preparing a cell lysate for cell-free protein synthesis 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 synthetase 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; and c) disrupting the harvested cells and preparing a cell lysate therefrom. Further aspects of the present invention relate in particular to eukaryotic cell lysates obtainable by the above methods as well as to a method for performing cell-free synthesis of a target protein comprising a non-canonical amino acid.

Dependencies are inferred from the claim text.