Means and methods for preparing target proteins manipulated by gene coding extensions in a selective manner for target proteins.

Abstract

To provide orthogonal translation systems which allow for the site-specific introduction of non-canonical amino acid residues into a target protein (POI).SOLUTION: The present invention relates to assembler fusion proteins, which bring an RNA-targeting polypeptide (RNA-TP) segment and an orthogonal aminoacyl tRNA synthetase (O-RS) segment into spatial proximity of one another, either by direct linkage in RNA-TP / O-RS fusion proteins, or though the action of "assemblers" fused to each of these segments in assembler fusion proteins (AFPs). The invention also relates to AFP combinations and nucleic acid molecules comprising a POI-encoding sequence together with a targeting nucleotide sequence that is able to interact with an RNA-TP. The invention further relates to nucleic acid molecules, expression cassettes and expression vectors and the like encoding the RNA-TP / O-RS fusion proteins or AFPs.SELECTED DRAWING: None

Claims

[Claim 1] Assembler fusion protein (AFP), (a) (a1) A polypeptide segment (IC-TP segment) derived from an intracellularly targeted polypeptide, wherein the intracellularly targeted polypeptide targets intracellular structural elements in or directly adjacent to the cytoplasm and is therefore locally concentrated, and (a2) A polypeptide segment (PSP segment) derived from a phase-separated polypeptide, wherein the phase-separated polypeptide has the ability to induce self-assembly in the cytoplasm of a cell, thereby creating sites of high local concentration in the cytoplasm. A first polypeptide segment that acts as an assembler (AP) selected from, (b) b1) RNA-targeted polypeptide (RNA-TP) segment, and b2) Orthogonal aminoacyl-tRNA synthetase (O-RS) segments, A second polypeptide segment (EP) that acts as an effector selected from, (c) A polypeptide segment (SYNZIP segment) derived from a SYNZIP polypeptide and having the ability to form a heterodimer coiled-coil protein structure, Includes, The polypeptide segments are functionally linked together on the AFP, forming an assembler fusion protein (AFP). [Claim 2] A combination of assembler fusion proteins (AFPs) comprising at least two different AFPs as described in claim 1, At least the first AFP includes an EP selected from the RNA-TP segment, At least the second AFP includes an EP selected from the O-RS segment, The polypeptide segments are functionally linked in each of the AFP polypeptide chains. At least one AFP of the above combination includes a first SYNZIP element, At least another AFP of the above combination includes a second SYNZIP element, The first SYNZIP element and the second SYNZIP element work together by forming a heterodimer structure. A combination of assembler fusion proteins. [Claim 3] The first SYNZIP element and the second SYNZIP element function together as a SYNZIP pair of heterospecific coiled coils. The SYNZIP pair is selected from the SYNZIP 1:2, SYNZIP 3:4, and SYNZIP 5:6 pairs. A combination of assembler fusion proteins according to claim 2. [Claim 4] An assembler fusion protein (AFP) according to claim 1, comprising at least two different EPs as defined in claim 1 within the same polypeptide chain, At least one EP is an RNA-TP segment, and at least one other EP is an O-RS segment. The polypeptide segment is functionally linked within the AFP, forming an assembler fusion protein. [Claim 5] (i) A nucleotide sequence encoding at least one AFP according to claim 1 or 4 or a combination of at least one AFP according to claim 2 or 3, or (ii) A nucleic acid sequence complementary to the nucleotide sequence of (i), or (iii)(i) and (ii), A nucleic acid molecule or a combination of two or more nucleic acid molecules containing [the specified substance]. [Claim 6] An expression cassette comprising a nucleotide sequence of a nucleic acid molecule or a combination of nucleic acid molecules as described in claim 5. [Claim 7] An expression vector comprising at least one expression cassette as described in claim 6. [Claim 8] At least one nucleic acid molecule or combination of nucleic acid molecules according to claim 5, At least one expression cassette according to claim 6, or At least one expression vector according to claim 7, Cells containing this substance. [Claim 9] (i) at least one EP selected from the RNA-TP segment, (ii) At least one EP selected from the O-RS segment, The cell according to claim 8, comprising a nucleotide sequence that encodes or is complementary to the nucleotide sequence encoding at least one AFP according to claim 1 or 4. [Claim 10] At least one of the two AFPs contains at least one RNA-TP segment, At least one of the two AFPs includes at least one O-RS segment, The cell according to claim 8, comprising a nucleotide sequence that codes for the combination described in claim 2 or 3, or a nucleotide sequence that is complementary to the nucleotide sequence that codes for it. [Claim 11] A method for preparing a target polypeptide (POI) containing one or more non-standard amino acid (ncAA) residues in its amino acid sequence, The method includes expressing POI by cells according to any one of claims 8 to 10 in the presence of one or more ncAAs, Cells: (i) A nucleotide sequence encoding a POI (CS) in which one or more ncAA residues of the POI are encoded by a selector codon (one or more) ＰＯＩ ); (ii) CS ＰＯＩ A targeted nucleotide sequence (TN) that is functionally linked to and capable of interacting with at least one RNA-TP segment of intracellular AFP; and (iii) CS ＰＯＩ One or more orthogonal tRNAs having an anticodon (one or more) complementary to the selector codon (one or more) of the specified tRNA. ｎｃＡＡ (O-tRNA ｎｃＡＡ ) molecules; including, The aforementioned O-tRNA ｎｃＡＡ The molecule combines with at least one or more O-RS segments of intracellular AFP to form one or more orthogonal O-RS / O-tRNAs. ｎｃＡＡ A method comprising forming pairs, which allow the introduction of one or more ncAA residues into the amino acid sequence of POI. [Claim 12] The method further includes recovering the expressed POI, The method according to claim 11. [Claim 13] (i) encoding a polypeptide of interest (POI), wherein the POI is encoded by a selector codon to one or more non-standard amino acid (ncAA) residues encoded on CS ＰＯＩ nucleotide sequence (CS ＰＯＩ ) and (ii) A nucleic acid molecule comprising a targeted nucleotide sequence (TN), wherein the RNA molecule containing the TN can interact with an RNA-targeted polypeptide (RNA-TP) via the TN, The RNA-TP is a nucleic acid molecule that is a component of the assembler fusion protein combination described in claim 2 or 3. [Claim 14] The kit is: - At least one ncAA or a salt thereof corresponding to at least one ncAA residue of POI, - comprising at least one expression vector as described in claim 7, A kit for preparing a target polypeptide (POI) having at least one non-standard amino acid (ncAA) residue.

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