Methods of RNA and protein synthesis
AssigneeUniversite De Nantes
InventorsVehary Sakanyan, Marina Snapyan, Anahit Ghochikyan, Francoise-Michele Lecocq
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Abstract
The present invention relates to methods for RNA and/or protein synthesis using in vitro or in vivo expression systems. More specifically, the present invention provides a method for RNA and/or protein synthesis characterized in that the concentration of alpha subunit of RNA polymerase, but not of other subunits, is increased in the cellular or cell-free system, comparing to its natural concentration existing in the cellular or cell-free system.
Claims27
A method for RNA or polypeptide synthesis from a DNA template comprising the steps of
- a) providing a cell-free system enabling RNA or polypeptide synthesis from a DNA template, said DNA template comprising a promoter with at least one UP element;
- b) recovering said synthesized RNA or polypeptide;
- characterized in that the concentration of α subunit of RNA polymerase, but not of other subunits, is increased in said cell-free system, comparing to its natural concentration existing in the cell-free system.
A method for the production of a protein from a DNA template in a cell-free system characterized in that it comprises the steps of
- a) providing in a reaction mixture, a bacterial cell-free system enabling the coupling of in vitro transcription of a specific gene from a DNA template, and the corresponding protein synthesis;
- b) adding to the reaction mixture the DNA template encoding the desired protein and purified α subunit of the RNA-polymerase;
- and,
- c) optionally, adding a thermostable RNA polymerase,
- d) recovering the produced protein.
A reaction mixture for cell-free protein synthesis characterized in that it is prepared from cells which overexpress the gene encoding α subunit of the RNA polymerase.
A reaction mixture for cell-free protein synthesis characterized in that it comprises a bacterial cell-free extract and an amount of purified α subunit of RNA polymerase.
A kit for cell-free RNA and/or protein synthesis characterized in that it comprises the following components:
- a) a cell-free extract, preferably E. coli S30 cell-free extract;
- b) purified α subunit of RNA polymerase;
- c) optionally, appropriate buffers and compounds for carrying out in vitro transcription and/or translation reaction;
- d) optionally, amino acid mixture lacking one amino acid.
A kit for cell-free RNA and/or protein synthesis characterized in that it comprises the following components:
- a) a cell-free extract, preferably E. coli S30 cell-free extract, wherein said cell-free extract is obtained from cells overexpressing subunit of RNA polymerase;
- b) optionally, appropriate buffers and compounds for carrying out in vitro transcription and/or translation reaction;
- c) optionally, amino acid mixture lacking one amino acid.
A purified α subunit of RNA polymerase for enhancing protein synthesis in a cell-free system.
Dependencies are inferred from the claim text.