Multimeric proteins of the peroxiredoxin family as scaffolding proteins

Abstract

The present invention relates to the use of a multimeric protein of the peroxiredoxin family as a scaffold protein, characterized in that one or more proteins of interest are linked to one or two N- and C-termini of one or more monomers of said peroxiredoxin, said peroxiredoxin having no redox activity.

Description

[Technical Field]

[0001] The present invention relates to the use of a multimeric protein derived from the peroxiredoxin (Prxs) family as a scaffold protein, characterized in that one or more target proteins are ligated to one or more N and C-terminuses of one or more monomers of the peroxiredoxin, and the peroxiredoxin does not have redox activity. [Background technology]

[0002] Enzymes are biocatalysts that can accelerate the synthesis, modification, or degradation of molecules, and they possess specific characteristics that allow them to function optimally under conditions compatible with living organisms. They are used in many fields, including the synthesis of bioplastics, biofuels, and therapeutic molecules.

[0003] Numerous studies have been conducted to improve the catalytic activity of enzymes. As a result, the rapid expansion of genetic engineering has made it possible to introduce activating mutations into specific enzymes. Advances in protein structure analysis and bioinformatics have made it possible to induce these mutations in a rational manner (Goldenzweig et al., 2016, Mol Cell. 2016 Jul 21;63(2):337-346).

[0004] Another approach involves incorporating reactants, intermediates, and enzymes into the same complex for localized concentration, thereby improving the efficiency of biochemical reactions. Various methods have been developed to enable the artificial assembly of enzymes. For example, direct fusion of enzymes has been used to modulate the expression and localization of two enzymes in resveratrol biosynthesis to enhance product formation within cells (Zhang et al., J. Am. Chem. Soc. 128:13030-13031 (2006)). Nevertheless, the structure of the fused enzymes may be adversely affected, potentially leading to enzyme inactivation.

[0005] Another approach involves immobilizing enzymes onto a proteinaceous support called a scaffold protein. Scaffold proteins allow for the localized concentration of one or more enzymes near a target substrate, making them particularly suitable for catalytic reactions involving macromolecular substrates. Furthermore, scaffold proteins can spatially bring enzymes belonging to the same metabolic pathway closer together, accelerating and simplifying the synthesis of small molecules.

[0006] Artificial multiprotein complexes derived from cellulosomes, in which enzymes are incorporated at specific sites, have been used to improve cellulose degradation (Fierobe et al. 2001. J. Biol. Chem. 276:21257-21261 and Fierobe et al. 2005, J Biol Chem. 280(16):16325-34). Furthermore, scaffold proteins utilizing protein-protein interaction domains have been developed to enhance the production efficiency of target molecules such as mevalonate and glucaric acid (Dueber et al. 2009. Nat. Biotechnol. 27:753-759).

[0007] Nevertheless, there remains a need to develop novel scaffold proteins that maintain stability under diverse environmental conditions while being strongly expressed within cells.

[0008] Peroxiredoxins (Prxs) are proteins found throughout the biological world, including in extremophiles. Peroxiredoxins are tolerant to a wide variety of conditions, including pH, temperature, and salt concentration. They acquire high stability to ensure essential redox functions for cell survival. These sequences contain a common motif surrounding a functionally important cysteine. The diversity of the sequences surrounding this motif confers appropriate physicochemical properties to the host organism. This protein family, in most cases, shares a common structure based on monomers of approximately 20 kDa that can multimerge to form decameric or dodecamer rings. This specific three-dimensional arrangement allows Prxs such as TSA1 from S. cerevisiae to exhibit floating ends located at the N-terminal and C-terminal positions of the monomer. [Overview of the project]

[0009] The inventors utilized the properties of peroxiredoxin, a multimeric protein of the peroxiredoxin family, specifically the TSA1 protein from Saccharomyces cerevisiae or peroxiredoxin from Pyrococcus furiosus, to develop a scaffold protein usable under diverse environmental conditions. Mutations were introduced to the active cysteine ​​to suppress the intrinsic redox activity of Prx and retain only its supportive function. The inventors demonstrated that the decameric structure of Prx remains conserved and the function of the target protein is maintained even when the target protein is ligated to the N and / or C-terminal position of the Prx monomer, indicating that peroxiredoxin is a scaffold protein with specific properties.

[0010] The present invention relates to the use of a multimeric protein derived from the peroxiredoxin family as a scaffold protein, characterized in that one or more target proteins are ligated to one or two N and C-terminuses of one or more monomers of the peroxiredoxin, and the peroxiredoxin has no redox activity. In one particular embodiment, the one or more target proteins are enzymes and the scaffold protein is used to enhance the catalytic activity of the enzymes, or the target proteins are proteins in the same signaling pathway and the scaffold protein is used to enhance the efficiency of the signaling pathway.

[0011] The peroxiredoxin is preferably peroxiredoxin TSA1 derived from Saccharomyces cerevisiae, as of Sequence ID No. 1, and more specifically, it contains cysteine ​​mutations at positions 48 and / or 171 of the Sequence ID No. 1 sequence, which are preferably substituted with alanine or serine, respectively.

[0012] In another preferred embodiment, the peroxiredoxin is a peroxiredoxin derived from Pyrococcus furiosus of SEQ ID NO: 16, more specifically, it contains cysteines mutated at positions 46 and / or 211 of the sequence of SEQ ID NO: 16, preferably substituted with alanine or serine respectively.

[0013] In a specific embodiment, the protein of interest is fused to one or two N- and C-termini of the peroxiredoxin monomer via a linker sequence.

[0014] In another specific embodiment, the peroxiredoxin monomer and the protein of interest are linked via an adapter / peptide ligand pair.

[0015] According to another aspect, the present invention relates to a multimeric protein derived from a family of peroxiredoxins, comprising at least one protein of interest linked to one or two N- and C-termini of one or more peroxiredoxin monomers having no redox activity, wherein the peroxiredoxin monomer and the protein of interest are linked via an adapter / peptide ligand pair. The present invention also relates to a multimeric protein derived from a family of peroxiredoxins, comprising at least two different proteins of interest linked to one or two N- and C-termini of one or more peroxiredoxin monomers having no redox activity. The peroxiredoxin is preferably peroxiredoxin TSA1 derived from Saccharomyces cerevisiae of SEQ ID NO: 1, more specifically, it contains cysteines mutated at positions 48 and / or 171 of the sequence of SEQ ID NO: 1, preferably substituted with alanine or serine respectively.

[0016] In another preferred embodiment, the peroxiredoxin is a peroxiredoxin derived from Pyrococcus furiosus of SEQ ID NO: 16, and more specifically, it contains cysteines mutated at positions 46 and / or 211 of the sequence of SEQ ID NO: 16, preferably substituted with alanine or serine respectively.

[0017] In a preferred embodiment, the peroxiredoxin monomer and the target protein are linked via an adapter / peptide ligand pair.

[0018] The present invention further relates to one or more nucleotide constructs encoding the above multimeric protein, and an expression vector containing the nucleotide construct. The present invention also relates to a host cell containing a multimeric protein, one or more nucleotide constructs, or an expression vector as described above.

Brief Description of Drawings

[0019] [Figure 1A] (A) From left to right, SDS-PAGE analysis of covalently grafted TSA1-CRDSAT, CRDSAT-TSA1, and CRDSAT-TSA1-(GGGS)3-CRDSAT. The target proteins indicated by the arrows were found in the ultrasonicated supernatant (SSo), lactose-sepharose resin (Lac-Seph), and elution fractions (elution 1-3). [Figure 1BC] (B) Size exclusion chromatography profiles obtained with the three structures shown in (A). (C) Fractions corresponding to the main peaks (P1 and P2) obtained in (B) are analyzed by SDS-PAGE. [Figure 1D] (D) Analysis of peak P1 obtained in (B) grafted with the ungrafted protein TSA1 by dynamic light scattering. The average size is reported in nanometers. [Figure 2]Analysis of the reconstitution of the 6xHIS-SPAG1-TPR3 complex with CRDSAT-TSA1-(GGGS)2 or 3-HSP90pep by SDS-PAGE. SSo and TALON represent the sonicated supernatant and affinity resin, respectively. [Figure 3] (A) DLS spectra (Dynamic Light Scattering) of free Pfu-PRX, PreScission 3C with scaffold, and (C) PETase with scaffold. The sizes of each of the major species are shown. [Figure 4] Thermograms recorded for Tsa1 (gray) and Pfu-Prx (black) obtained by isothermal titration microcalorimetry at 25°C. [Figure 5] The denaturation curve for Pfu-Prx obtained by differential scanning microcalorimetry. The semi-denaturation temperature measured at the peak of each peak is shown. [Figure 6] Monitoring of the degradation of 6His-SPAG1-TPR3 by protease 3C using Pfu-Prx as a scaffold by electrophoresis on an SDS-modified gel. The lane labeled "Pfu-Prx:3C" corresponds to the protease with a scaffold. The lane labeled "Substrate" corresponds to 6His-SPAG1-Cter after 60 minutes at 10°C in the absence of protease. The lanes labeled 1 to 60 correspond to the deposition of reaction mixture samples collected at 1, 2, 5, 10, 15, 30, and 60 minutes. The lane labeled "MT" corresponds to the size marker (MT) expressed in kDa. [Figure 7] Monitoring of the pH of the reaction solution during PET degradation by PETase using Pfu-Prx as a scaffold. The black curve corresponds to PET in the presence of the enzyme. The gray curve corresponds to the control without the enzyme. [Modes for carrying out the invention]

[0020] The inventors have shown that a peroxiredoxin multimer protein, in which the free N and C-terminuses of the monomer are linked to the target protein, maintains its specific three-dimensional structure as a decamer or dodecamer and preserves the function of the target protein. Therefore, peroxiredoxin multimer proteins can be used as scaffold proteins.

[0021] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to chains of amino acids linked by peptide bonds, regardless of the number of amino acids that make up the chain.

[0022] Scaffold proteins are proteins that enable the linking of multiple identical or different target proteins within a single protein complex, in order to facilitate interactions and functions between various target proteins, or to locally assemble target proteins.

[0023] Scaffold proteins allow compounds in the same metabolic pathway to assemble into a single complex by linking multiple target proteins, for example, by limiting unwanted interactions and increasing the proximity and concentration of the target proteins, thereby improving the efficiency of enzymatic reactions. Scaffold proteins can also localize target proteins to specific regions of the cell to enhance their function.

[0024] Peroxiredoxin protein (Prx or PRDX) (EC 1.11.1.24), also known as TSA (thiol-specific antioxidant), is a highly conserved enzyme with peroxidase activity due to a cysteine ​​residue called peroxidatic cysteine ​​(Cp). Peroxiredoxin reduces peroxides via its oxidation site. Subsequently, Cp cysteine ​​"resolves" cysteine ​​(C R) reacts with the electron donor to form a disulfide bridge, which is then reduced by the electron donor to complete the catalytic cycle (Revue Rhee SG, Mol Cells. 2016. 39(1):1-5).

[0025] Peroxiredoxin is C R Depending on whether or not it contains cysteine, it can be classified into subclasses of 2-Cys, atypical 2-Cys, and 1-Cys peroxiredoxins. In the atypical 2-Cys subclass of Prx, C R Cysteine ​​is located in an atypical position.

[0026] Recently, peroxiredoxins have also been classified into six classes called AhpC-Prx1, BCP-PrxQ, Prx5, Prx6, Tpx, and AhpE (Soito, Laura et al. 2011, Nucleic Acids Res. England. 39 (Database issue):D332-7.doi:10.1093 / nar / gkq1060).

[0027] Members of subclasses AhpC / Prx1 and Prx6 combine with each other to form octamers, decamers, or dodecamers (Hall et al. 2010, J Mol Biol. 402(1):194-209; Parsonage et al. 2005, Biochemistry, 2005;44(31):10583-92).

[0028] The peroxiredoxin of this patent application can be polymerized to preferably form a decamer or dodecamer, more specifically a peroxiredoxin monomer of the AhpC-Prx1 or Prx6 subclass. The Prx monomer is preferably derived from the AhpC-Prx1 subclass.

[0029] The subclass AhpC-Prx1 consists essentially of "typical 2-Cys" Prx. Members of this subclass include bacterial AhpC proteins, tryparedoxin peroxidase, plant Prx including Arabidopsis thaliana-derived 2-Cys Pxrs and barley-derived Bas1 Pxrs, yeast proteins TSA1 and TSA2, as well as human Prx I, II, III, and IV. In addition to the common basic structure of Prxs, members of this subclass are C R It has a C-terminal extension that includes a B-type interface, which forms a disulfide bond with the Cp of its partner subunit (Hall et al 2010, J Mol Biol. 402(1):194-209).

[0030] The subclass Prx6 includes bacterial Prx6 proteins, 1-Cys plant Prx proteins from Arabidopsis thaliana and barley, mitochondrial yeast protein Prx1, and human PrxVI. Prx6 proteins are closely related to the AhpC / Prx1 subclass. Prx6 proteins involve C-terminal elongation, form type B dimers, and in some cases, higher-order oligomeric states. However, in contrast to the AhpC-Prx1 subclass, while representative 2-Cys proteins exist, the members of the Prx6 subclass are primarily 1-Cys (Deponte and Becker 2005).

[0031] [Table 1A]

[0032] [Table 1B]

[0033] [Table 1C]

[0034] [Table 1D]

[0035] Table 1E

[0036] Preferably, monomers of multimeric proteins derived from the peroxiredoxin family are selected from the following group: peroxiredoxin TSA1 from Saccharomyces cerevisiae (SEQ ID NO: 1), peroxiredoxin from Aeropyrum pernix K1 (SEQ ID NO: 2), triparedoxin peroxidase from Crithidia fasciculata (SEQ ID NO: 3), peroxiredoxin from Chlamydomonas reinhardtii (SEQ ID NO: 4), chloroplastic 2-Cys peroxiredoxin BAS1 from Arabidopsis thaliana (SEQ ID NO: 5), mitochondrial peroxiredoxin from Leishmania infantum (SEQ ID NO: 6), peroxiredoxin-1 from rat (Rattus norvegicus) (SEQ ID NO: 7), Leishmania Tryparedoxin peroxidase from major (SEQ ID NO: 8), peroxiredoxin-4 (isoform 1) from Homo sapiens (SEQ ID NO: 9), peroxiredoxin TSA2 from Saccharomyces cerevisiae (SEQ ID NO: 10), alkylhydroperoxide reductase C from Helicobacter pylori (SEQ ID NO: 11), peroxiredoxin-2 (isoform 1) from Homo sapiens (SEQ ID NO: 12), alkylhydroperoxide reductase C from Salmonella typhimurium (SEQ ID NO: 13), thioredoxin peroxidase from Schistosoma mansoni (blood fluke) (SEQ ID NO: 14), Trypanosoma Presumed tryparedoxin peroxidase from cruzi (SEQ ID NO: 15), peroxiredoxin from Pyrococcus furiosus (SEQ ID NO: 16), PyrococcusPeroxiredoxin from *Horikoshii* (SEQ ID NO: 17), peroxiredoxin-4 from *Mus musculus* (SEQ ID NO: 18), alkylhydroperoxide reductase C from *Amphibacillus xylanus* (SEQ ID NO: 19), peroxiredoxin-1 from *Ancylostoma ceylanicum* (SEQ ID NO: 20), alkylhydroperoxide reductase C from *Enterococcus faecalis* (SEQ ID NO: 21), peroxiredoxin-4 from *Larimichthys crocea* (SEQ ID NO: 22), peroxiredoxin from *Sulfolobus islandicus* (SEQ ID NO: 23), presumed 2-Cys peroxiredoxin from *Plasmodium vivax* (SEQ ID NO: 24), peroxiredoxin from *Leishmania braziliensis* (SEQ ID NO: 25), peroxiredoxin from *Akkermansia muciniphila* (SEQ ID NO: 26), *Escherichia coli* Alkyl hydroperoxide reductase C derived from coli (SEQ ID NO: 27) and alkyl hydroperoxide reductase C derived from Mycobacterium tuberculosis H37Rv (SEQ ID NO: 28), or functional variants thereof.

[0037] "Peroxiredoxin monomer" or "peroxiredoxin" refers to any monomer of natural peroxiredoxin, but more specifically, to its functional variants that retain the ability to multimerge to form a decameric or dodecameric ring, in which the N and C-terminuses of the monomer are exposed outside this ring.

[0038] Preferably, as used herein, the terms “mutant” or “functional mutant” refer to a polypeptide having an amino acid sequence that exhibits at least 70, 75, 80, 85, 90, 95, or 99% sequence identity with the amino acid sequence in peroxiredoxin described above, more specifically, one of the sequences selected from the group consisting of SEQ ID NOs: 1 to 28, and which retains the ability to multimerize to form a decameric or dodecamer ring, with the N and C-terminuses of the monomer exposed outside this ring.

[0039] In this specification, the terms “sequence identity” or “identity” refer to the number (%) of positional matches (identical amino acid residues) resulting from the alignment of two polypeptide sequences. Sequence identity is determined by comparing sequences when they are aligned to maximize overlap and identity while minimizing gaps between sequences. More specifically, sequence identity can be determined using a predetermined number of global or local mathematical alignment algorithms, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman and Wunsch's algorithm; Needleman and Wunsch, 1970) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith and Waterman's algorithm (Smith and Waterman, 1981) or Altschul's algorithm (Altschul et al, 1997; Altschul et al, 2005)). Alignment for the purpose of determining the percentage of amino acid sequence identity can be performed in a variety of ways within the scope of the skills of those skilled in the art, such as using digital software accessible to the public on websites like http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . Experts in this field can determine appropriate parameters for measuring alignment, including algorithms necessary to obtain the maximum alignment over the entire length of the sequences being compared. For the purposes of this document, the percentage value of amino acid sequence identity refers to the value generated using the EMBOSS Needle pairwise sequence alignment program.This program uses the Needleman-Wunsch algorithm to create the optimal global alignment of two sequences, with all search parameters adjusted to their default values: scoring matrix = BLOSUM62, open spaces = 10, extended spaces = 0.5, end space penalty = false, end open spaces = 10, and end extended spaces = 0.5.

[0040] The terms "mutant" or "functional mutant" preferably refer to polypeptides having an amino acid sequence different from the sequence of sequence numbers 1 to 28 in one or more conserved substitutions.

[0041] In this invention, "substituted" or "modified" means an amino acid that has been altered or modified from a natural amino acid.

[0042] As used herein, the term “conservative substitution” means the substitution of one amino acid residue with another amino acid residue without adversely affecting the three-dimensional structure and overall function of a protein, and this includes, but is not limited to, the substitution of one amino acid with another amino acid having similar properties (e.g., polarity, hydrogen bonding potential, acidity, basic acidity, shape, hydrophobicity, aromaticity, etc.).

[0043] Examples of conservative substitutions are found in the groups of basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (methionine, leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and minor amino acids (glycine, alanine, serine, and threonine).

[0044] In this patent application, peroxiredoxin or a functional variant of peroxiredoxin is peroxiredoxin that does not exhibit redox activity. More specifically, peroxiredoxin or a peroxiredoxin variant contains a mutated peroxidatic cysteine ​​and / or a “degradation” residue. The peroxidatic cysteine ​​and “degradation” cysteine ​​of peroxiredoxin are characterized for a number of peroxiredoxin proteins and are well known to those skilled in the art (Nelson et al. Proteins, 2012, 79(3):947-964; http: / / csb.wfu.edu / prex / search.php). More specifically, C R This is conserved at the same position as C172 in human PrxII. As an example, Table 1 shows the peroxidase cysteine ​​and degraded cysteine ​​of numerous peroxiredoxin proteins.

[0045] The peroxiredoxin monomer described above preferably has a peroxidatic and / or "degraded" cysteine ​​that has been mutated by another amino acid, preferably alanine or serine.

[0046] This patent application preferably relates to the peroxiredoxin monomer TSA1 derived from Saccharomyces cerevisiae, and more preferably to a mutant Prx monomer having a cysteine ​​mutation at positions 48 and / or 171 of the sequence of SEQ ID NO: 1, preferably with any other amino acid, preferably alanine or serine, and more preferably containing or consisting of the sequence of SEQ ID NO: 29.

[0047] The present inventors have demonstrated that peroxiredoxin derived from Pyrococcus furiosus is particularly stable, retaining its decaper structure even at low concentrations, and is particularly advantageous as a scaffold protein. Therefore, in another preferred embodiment, this patent application relates to a peroxiredoxin monomer derived from Pyrococcus furiosus, preferably a mutant Prx monomer having a cysteine ​​mutated by any other amino acid, preferably alanine or serine, at positions 46 and / or 211 of the sequence of SEQ ID NO: 16, and preferably comprising or consisting of the sequence of SEQ ID NO: 52.

[0048] Peroxiredoxins, which can multimerize to form decameric or dodecameric rings, have floating ends at the N and C-terminal positions of each monomer. Therefore, as described above, one or more target proteins can be ligated to one or more N and C-terminuses of one or more monomers of peroxiredoxin.

[0049] The peroxiredoxin family-derived multimeric protein according to the present invention preferably comprises at least two different target proteins ligated to the N and C-terminuses of one or more peroxiredoxin monomers that do not have redox activity.

[0050] The target protein comprises any protein whose activity or function is enhanced when assembled into a protein complex. The target protein preferably has a size of less than 50 kDa, preferably less than 40, 30, 20, or 10 kDa, and preferably less than 20 kDa.

[0051] The target protein according to the present invention is preferably an enzyme or protein in a metabolic pathway.

[0052] In a particular embodiment, the target protein is a protein from the same metabolic pathway, and the scaffold protein is used to enhance the efficiency of synthesis. For example, proteins from the same metabolic pathway are proteins that enable the synthesis of target molecules such as catechin, D-glucaric acid, H2, hydroquinone, resveratrol, butyrate, γ-aminobutyric acid, and mevalonate, (2S,5S)-hexanediol, mono(2-hydroxyethyl) terephthalate (MHET), terephthalic acid, and ethylene glycol.

[0053] In another specific embodiment, the protein of interest is an enzyme, and the scaffold protein is used to enhance the catalytic activity of the enzyme. The enzyme is preferably an enzyme having a polymer substrate. For example, the enzyme may be selected from the group consisting of: PET hydrolase (PETase), lysozyme, MHETase, alcohol dehydrogenase, lytic polysaccharide monooxygenases (LPMOs), and endopeptidases.

[0054] In certain cases, the target protein is not the reporter protein.

[0055] The term "reporter protein" refers to a protein that has characteristics that allow it to be observed or quantified, for example, by bioluminescence (e.g., fluorescence), detection of enzyme activity, or recognition by an antibody. Therefore, a reporter protein may be selected from a fluorescent protein, an enzyme that has the effect of causing the appearance of a colored product or an easily characterized phenotype, or any other protein with other quantifiable activity, or a short sequence of amino acids called a tag. Reporter proteins are selected from luciferase, β-galactosidase (LacZ) or hydrolase (β-glucuronidase, alkaline phosphatase), chloramphenicol acetyltransferase (CAT) and β-lactamase (TEM-1), tag HA (human influenza hemagglutinin), FLAG, polyHis6, and fluorescent proteins.

[0056] The target protein can be linked to the N-terminus or C-terminus of the peroxiredoxin monomer by covalent or non-covalent bonds.

[0057] In one particular embodiment, the protein of interest is covalently fused to the N-terminus or C-terminus of the peroxiredoxin monomer.

[0058] As used in this patent application, the term "fusion protein" refers to a protein comprising at least two different polypeptides that do not originate from the same protein.

[0059] The two proteins may be fused directly or via a peptide sequence called a linker, which can attach various proteins or protein fragments to the proteins so that they adopt a better three-dimensional structure for protein activity. More specifically, the nucleotide sequence encodes a peptide sequence of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 amino acids. In one preferred embodiment, the linker encodes a peptide sequence consisting of GGGS, GGGSGGGS (SEQ ID NO: 30), or GGGSGGGSGGGS (SEQ ID NO: 31).

[0060] In another specific embodiment, the protein of interest is non-covalently linked to the N-terminus or C-terminus of a peroxiredoxin monomer, preferably via an adapter / peptide ligand pair. The adapter / peptide ligand pair consists of an adapter region of a protein having high affinity for a peptide ligand having a specific sequence motif that enables effective coupling of the two linked proteins.

[0061] Using adapter / peptide ligand pairs allows for control over the number and type of target proteins ligated to the Prx monomer of the scaffolding protein, thereby preventing steric hindrance of the target proteins on the scaffolding protein.

[0062] Adapter / peptide ligand pairs that can be used in the context of the present invention are well known to those skilled in the art, and include, for example, domains SH3, SH2, PDZ, and GBD (GTPase-binding domain) (Horn AHC et al. 2015, Front Bioeng Biotechnol. 3:191).

[0063] The inventors also characterized novel adapter / peptide-ligand pairs containing the protein linking domains of Rsa1 (Uniprot ID: Q08932), NUFIP1 (Uniprot ID: Q9UHK0), HSP90 (Uniprot ID: P08238, P07900), HSP70 (Uniprot ID: P0DMV8), RPAP3 (Uniprot: Q9H6T3), SPAG1 (Uniprot ID: Q07617), or Tah1 (Uniprot ID: P25638).

[0064] In one preferred embodiment, the adapter / peptide ligand pair is selected from the following pairs (see Table 2): -HSP90C of sequence number 32 or 33 and SPAG1-622742 of sequence number 34; -HSP90C (sequence number 32 or 33) and SPAG1-206327opt (sequence number 35); - HSP90C (sequence number 32 or 33) and RPAP3-281396 (sequence number 36); - HSP90C with sequence number 32 or 33 and Tah1 with sequence number 37; -HSP70C (SEQ ID NO: 38) and SPAG1-622742 (SEQ ID NO: 34); -HSP70C (SEQ ID NO: 38) and SPAG1-206327opt (SEQ ID NO: 35); - ySNU13 in sequence number 39 and Rsa1-238259 in sequence number 40; -hSNU13 (sequence number 41) and NUFIP1-233255 (sequence number 42); -Tah1-93111 in SEQ ID NO: 43 and Pih1-258344 in SEQ ID NO: 44; - RPAP3-396455 (sequence number 45) and PIH1D1-199290 (sequence number 46); - ZNHIT3-85155 (SEQ ID NO: 47) and NUFIP1-462495 (SEQ ID NO: 48); -Sequence ID 49 Hit1-70164 and Sequence ID 50 Rsa1-317352.

[0065] [Table 2]

[0066] More specifically, the adapter domain and / or peptide ligand described above is fused to the peroxiredoxin monomer, preferably one or two N or C-terminuses, and the corresponding adapter domain and / or peptide ligand is fused to the target protein such that the peroxiredoxin monomer and the target protein are linked to each other via the linkage of the adapter / peptide ligand pair.

[0067] In one preferred embodiment, the protein is synthesized using recombinant technology. In this case, a nucleic acid construct containing, or comprising, the nucleic acid sequence encoding the above-mentioned protein is used and expressed in a host cell. The nucleic acids and expression vectors that may be used to produce the protein according to this patent application are described below.

[0068] Nucleic acids and expression vectors This patent application further relates to one or more nucleotide constructs encoding the aforementioned multimeric proteins.

[0069] The terms “nucleic acid” or “nucleotide sequence” may be used interchangeably to refer to any molecule composed of or containing nucleic acids. Nucleic acids may be oligonucleotides or polynucleotides. Nucleic acid sequences may be DNA or RNA. Nucleic acid sequences may be chemically or artificially modified. Nucleic acid sequences may include nucleic acids, peptides, morpholinos, blocked nucleic acids, as well as glycol nucleic acids and threose nucleic acids. Each of these sequences has a different skeletal structure than natural DNA or RNA. Phosphothioate nucleotides may be used. Other nucleic acid analogs such as methylphosphonates, phosphoramidates, phosphorodithioates, N3'P5'-phosphoramidates and phosphorothioate oligoribonucleotides, as well as analogs 2'-O-allyl- and 2'-O-methylribonucleotide methylphosphonates, may be used in the context of the present invention.

[0070] In a particular embodiment, this patent application relates to a nucleotide construct encoding a peroxiredoxin monomer fused to a protein of interest, preferably via a linker, at one or two N and C-terminuses.

[0071] The nucleotide sequence encoding the peroxiredoxin monomer described above is preferably fused to the nucleotide sequence encoding the protein of interest via a "linker," preferably a nucleotide sequence encoding a peptide sequence that enables the linking of various proteins or protein fragments so that the protein can conform better for protein activity. More specifically, the nucleotide sequence encodes a peptide sequence of 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50 amino acids. In one preferred embodiment, the linker encodes the peptide sequence GGSGGS (SEQ ID NO: 30), GGGSGGGSGGS (SEQ ID NO: 31), or GGGS.

[0072] Fusion of two or more nucleotide sequences refers to a combination of two or more nucleotide sequences that encode different proteins or protein fragments, and translation of the two fused genes yields a single functional polypeptide. In the context of this invention, the peroxiredoxin monomer fuses with the target protein to form a multimeric "scaffold" protein that enables the complexation of the target protein or multiple target proteins.

[0073] In another specific embodiment, the present patent application relates to nucleotide constructs encoding a peroxiredoxin monomer fused at one or two N and C-terminuses to a first peptide sequence of the adapter domain defined above, as well as nucleotide constructs encoding a protein of interest fused to a corresponding peptide ligand of an adapter / peptide-ligand pair. The peroxiredoxin monomer can non-covalently link to one or more proteins of interest via the adapter / peptide-ligand pair to form a multimeric "scaffold" protein that complexes one or more proteins of interest.

[0074] The nucleotide sequences described preferably refer to complementary DNA (also called cDNA) encoding a peroxiredoxin monomer or a protein of interest. The nucleotide constructs of this patent application preferably include sequences encoding a peroxiredoxin monomer as described above, and preferably include the sequence of Sequence ID No. 51 encoding a peroxiredoxin monomer TSA1 derived from S. cerevisiae with mutations at positions 48 and 171.

[0075] In another preferred embodiment, the nucleotide construct of the present patent application comprises the sequence of SEQ ID NO: 53, which encodes a peroxiredoxin monomer derived from Pyrococcus furiosus with mutations at positions 46 and 211.

[0076] This patent application further relates to a vector comprising the above-mentioned nucleotide construct.

[0077] Here, "vector" refers to a DNA molecule that can have either a single-stranded or double-stranded form.

[0078] The recombinant vector according to the present invention is preferably a plasmid vector or an integration vector. In one embodiment of the present invention, the vector is a plasmid. As used herein, “plasmid” means a double-stranded circular DNA molecule having an origin of replication that can autonomously replicate within a cell and a select gene that is not lost by the organism during cell proliferation. Numerous vectors are known; the selection of an appropriate vector depends on the intended use of the vector (e.g., replication of a target sequence, expression of this sequence, maintenance of this sequence in an extrachromosomal form, or integration into the host chromosomal apparatus) and the properties of the host cell.

[0079] The vector is preferably an expression vector containing all the elements necessary for the expression of the target gene as defined above. For example, the vector includes an expression cassette containing at least one target gene as defined above, under the control of appropriate sequences (promoter, activator, intron, start codon (ATG), codon stop, polyadenylation signal, splicing site) that regulate transcription and, optionally, translation.

[0080] The vector is preferably a DNA plasmid vector. In one particular embodiment, the vector is adapted for use in prokaryotic host cells and is preferably selected from: ACYC184, pBeloBacll, pBR332, pBAD33, pBBR1MCS and its derivatives, pSC101, SuperCos (cosmid), pWE15 (cosmid), pTrc99A, pBAD24, vectors containing ColE1 origin and its derivatives, pUC, pBluescript, pCARGHO, pET, pGEM, pnEA, pnYK, pnCS, and pTZ vectors.

[0081] In addition to the coding region, the gene of interest may include, or be combined with, one or more elements that promote or enhance the expression of the gene, such as an activation sequence, a response element, an insulator, a polyadenylation signal, and / or any other functional element.

[0082] The peroxiredoxin monomer and / or the nucleotide sequence encoding the protein of interest can be inserted into an expression vector under the transcriptional control of a promoter to enable protein expression.

[0083] A "promoter" is a polynucleotide that can positively control the expression of an operationally linked nucleotide sequence within a cell. Promoters, or promoter sequences, are located near genes and are essential DNA regions for transcription from DNA to RNA. Promoter sequences are generally located upstream of the transcription start site. The promoter sequence corresponds to the region to which RNA polymerase first attaches before starting RNA synthesis.

[0084] host cell This patent application further relates to host cells comprising, all of the above-mentioned multimeric proteins, nucleotide constructs, or vectors. The host cell can be either a eukaryotic or prokaryotic cell.

[0085] Eukaryotic cells include, in particular, animal cells, fungal cells, insect cells, plant cells, and algal cells. Eukaryotic host cells are preferably selected from the group consisting of: Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, and Pichia species. sp.), Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorphs, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp.), Fusarium gramineum, Fusarium venenatum, Neurospora crassa, Spodoptera frugiperda Sf9, Spodoptera frugiperda Sf21, Chlamydomonas reinhardtii.

[0086] The prokaryotic cells are preferably selected from the group consisting of Escherichia coli, Lactobacillus sp., Salmonella sp., Shigella sp., Rhodococcus sp., Bacillus sp., and Pseudomonas sp.

[0087] Use of multimeric proteins The multimeric proteins derived from the peroxiredoxin family mentioned above are used as scaffold proteins. Scaffold proteins enable the complexation of one or more target proteins to improve the function of one or more target proteins.

[0088] Scaffold proteins preferably enable the enhancement of the synthesis of the target molecule by complexing one or more metabolic pathway proteins with the scaffold protein. Functional enhancement, when the target protein is a metabolic pathway protein, means enhancing the synthesis of the target molecule corresponding to the final or intermediate product of the metabolic pathway. More specifically, the use of scaffold proteins according to the present invention enables an enhancement of the metabolic production level of the target molecule by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 times compared to production in the presence of free enzymes.

[0089] The target molecules are not limited to, but include catechin, D-glucaric acid, H2, hydroquinone, resveratrol, butyrate, γ-aminobutyric acid, mevalonate, (2S,5S) hexanediol, mono(2-hydroxyethyl) terephthalate (MHET), terephthalic acid, and ethylene glycol.

[0090] When the target protein is an enzyme, improving its function means enhancing its catalytic activity. Therefore, scaffold proteins can increase the enzyme's concentration and stability, thereby increasing its catalytic activity.

[0091] In a particular embodiment, the protein of interest is a hydrolase, preferably a hydrolase having a high molecular weight substrate. The hydrolase is preferably selected from the following: PET hydrolase, MHETase, lysozyme, alcohol dehydrogenase, LPMO, and endopeptidase.

[0092] The present invention will be better understood by referring to the following examples, but these examples are not intended to impose any limitations and are merely to document specific embodiments and particular advantageous characteristics of the present invention. [Examples]

[0093] 1. Example of a covalent graft In the following example, human galectin-3 CRD SAT The domain was covalently grafted onto C171A, a variant of the TSA1 protein derived from S. cerevisiae. This resulted in the synthesis of a plasmid suitable for protein overexpression in E. coli. (CRD) SAT The oligonucleotide sequence of the domain (Kriznik et al., 2019, Biotechnol.J., doi:10.1002 / biot.201800214) was positioned upstream (Nter position), downstream (Cter position), or on both sides of the TSA1-C171A oligonucleotide sequence on these plasmids. When positioned at the Cter position, CRDSAT Prior to the array, there is a non-natural linker region of the protein sequence GGGSGGGSGGGS (i.e., (GGGS)3, SEQ ID NO: 31).

[0094] a. Protocol Each plasmid was transformed into competent Escherichia coli BL21(DE3)pRARE2 Ca 2+The strain was introduced by a heat shock at 42°C for 60 seconds. Transformed clones were selected on solid LB-agar medium supplemented with ampicillin (A) and chloramphenicol (C). After incubation at 37°C overnight, isolated colonies were cultured in 50 mL of LB-AC medium at 37°C for 16 hours with stirring. 10 mL of this culture was added to 500 mL of LB-AC medium and cultured at 37°C with stirring until the absorbance of the solution, measured at a wavelength of 600 nm, was 0.6. Overexpression of the recombinant protein encoded by the plasmid was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG, final concentration 0.25 mM). After stirring the culture at 20°C for 16 hours, it was centrifuged at 4000 g for 45 minutes. The bacterial pellet is placed in 20 mL of Buffer 1 (25 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM TCEP) and dissolved by sonication. The sonicated material is centrifuged at 20,000 g at 4°C for 30 minutes, and the supernatant is incubated with 1 mL of 50% lactose-Sepharose resin pre-equalized with Buffer 1 at 4°C for 30 minutes. The resin is washed three times with Buffer 1. The recombinant protein is eluted by adding 1.5 mL of Buffer 1 containing 200 mM D-lactose three times. Samples are taken at each purification step (dissolution, fixation, elution) and analyzed by SDS-PAGE. If necessary, the fraction containing the target protein is collected and concentrated to a volume of 0.25 mL. This solution is injected into a Superose 6 Increase analytical column (S6I, GE Healthcare) pre-equalized with Buffer 2 (20 mM HEPES, pH 7.5, 100 mM NaCl). Proteins are eluted with buffer 2 according to their size. The eluate is monitored by measuring the absorbance at 280 nm. A 0.5 mL fraction is collected at the column outlet. The presence of the target protein in the fraction is monitored by SDS-PAGE.

[0095] b.Results Protein CRD was found in the sonication supernatant, on the lactose-cepharose resin, and in the eluted fraction from the lactose-cepharose resin. SAT -TSA1, TSA1-CRDSAT , CRD SAT -TSA1-CRD SAT The presence of CRD was detected (Figure 1A). SDS-PAGE analysis of most of the peaks in each eluate revealed that these peak fractions contained CRD. SAT -TSA1, TSA1-CRD SAT , or CRD SAT -TSA1-CRD SAT The protein was shown to be present in high purity. The retention amount on the S6I column and the protein migration profile on SDS-PAGE were compared with the protein CRD. SAT -TSA1, TSA1-CRD SAT , or CRD SAT -TSA1-CRD SAT This is consistent with the molecular weight (Figures 1B and 1C). Dynamic light scattering (DLS) analysis of the S6I fraction containing the target protein was performed at 20°C using a NanoSizer instrument (Malvern). This analysis shows monodisperse peaks for each target protein that very well characterize most species in solution. Protein CRD SAT -TSA1, TSA1-CRD SAT , CRD SAT -TSA1-CRD SAT The size is Nter, Cter, or Nter / Cter CRD SAT It fits the domain-decorated TSA1 decamer (Figure 1D).

[0096] 2. Example of a non-covalent graft a. Protocol For the production of recombinant protein, we designed a pCARGHO2-type plasmid encoding the oligonucleotide sequence of protein TSA1-C171A, followed by a GGGSGGGS linker (SEQ ID NO: 30) or a GGGSGGGSGGGS linker (SEQ ID NO: 31), and then the peptide sequence DASRMEEVD (SEQ ID NO: 32) obtained from human protein HSP90 (HSP90pep). The pnEA vector can encode the TPR3 domain of human protein SPAG1 (corresponding to the 622-742 region of the protein, SEQ ID NO: 34), and is fused with a 6-histidine (6xHIS) label at the N-terminus. As detailed above, recombinant protein CRD in LB medium. SAT -TSA1-(GGGS) 2または3 -Produce a 30 mL culture of E. coli BL21(DE3)pRARE2 strain overexpressing HSP90pep and 6xHIS-SPAG1-TPR3.

[0097] Pellet 30 mL of bacterial culture containing expressed 6xHIS-SPAG1-TPR3, incorporate into 1.5 mL of Buffer 3 (25 mM HEPES, pH 7.5, 300 mM NaCl, 10 mM imidazole, 0.5 mM TCEP), and centrifuge at 16,000 g for 20 minutes. Contact 500 μL of supernatant with 150 μL of TALON resin suspension at 4°C for 20 minutes. Wash the resin three times with 500 μL of Buffer 1. CRD SAT Add 30 mL of bacterial culture containing -TSA1-(GGGS)2 or 3-HSP90pep to 1.5 mL of Buffer 1 and centrifuge at 16,000 g for 20 minutes. Contact 500 μL of supernatant with TALON beads immobilized with 6xHIS-SPAG1-TPR3 at 4°C for 30 minutes. Wash the resin three times with 500 μL of Buffer 3. Elute the protein immobilized on the beads with 150 μL of Buffer 3 mixed with 300 mM imidazole. Elute 150 μL of TALON beads with CRD. SATThe degree of nonspecific fixation of proteins without 6xHIS labeling to resin can be evaluated by directly contacting a control (treated under conditions exactly the same as those used above) with 500 μL of supernatant containing -TSA1-(GGGS)2 or 3-HSP90pep.

[0098] b.Results CRD SAT The reconstitution of the complex of -TSA1-(GGGS)2 or 3-HSP90pep and 6xHIS-SPAG1-TPR3 was monitored by SDS-PAGE (Figure 2). The inventors observed that the target protein was present in the sonication supernatant and that the immobilization of 6xHIS-SPAG1-TPR3 to TALON beads was specific. By comparison with control experiments, the inventors found that the protein CRD SAT This study showed that -TSA1-(GGGS)2 or 3-HSP90pep is more readily retained on TALON beads immobilized with 6xHIS-SPAG1-TPR3 than on bare TALON beads. Protein CRD SAT -TSA1-(GGGS)2 or 3-HSP90pep and 6xHIS-SPAG1-TPR3 are co-eluted stoichiometrically upon addition of high concentrations of imidazole. The small amount of protein eluted in the control experiment indicates CRD. SAT -TSA1-(GGGS) 2または3 - This suggests that a specific complex of HSP90pep and 6xHIS-SPAG1-TPR3 is formed in solution.

[0099] CRD in E. coli BL21(DE3) strain SAT -TSA1-(GGGS) 2または3 The non-covalent complex is reconstituted by co-expressing HSP90pep and 6xHIS-SPAG1-TPR3. Therefore, the protein-coding plasmids must have compatible origins of replication. Each plasmid confers a single antibiotic resistance to select bacteria co-transformed by the two plasmids. The non-covalent complex is purified by one or other labels, preferably by 6xHIS labeling in the above case.

[0100] 3. Example of a target enzyme graft a. Equipment Production and purification of recombinant proteins The enzymes intended for peroxiredoxin-type scaffolding and grafting are heterologously produced in E. coli BL21(DE3)pRARE2 using an inducible expression plasmid. The proteins are purified using two sequential chromatography steps, starting from the soluble fraction of the cell extract in 25 mM HEPES (pH 7.5) and 150 mM NaCl buffer. The first step is metal affinity chromatography (GE Healthcare HiTrap Talon Crude 5 mL), followed by size exclusion chromatography (Superose® 6 Increase 10 / 300 GL).

[0101] Assembly of the enzyme scaffold The selected scaffold corresponds to Prx1 (Pfu-Prx) from Pyrococcus furiosus, in which a cysteine ​​residue is mutated to a serine residue (SEQ ID NO: 52). The selected target enzymes are the protease PreScission 3C and PET hydrolase (or PETase) obtained from compost (SEQ ID NOs: 54 and 55). Enzyme assembly on the scaffold is produced by gene fusion or by using the protein / adapter peptide pairs described above. A polyglycine-type linker separates the target enzyme from the scaffold.

[0102] b.Results Analysis of protein particle size by dynamic light scattering (DLS) DLS experiments were performed using a Zetasizer Nano ZS instrument (Malvern Panalytical) and a small-volume quartz cell in 25 mM HEPES (pH 7.5) and 150 mM NaCl buffer at 20°C. The results are shown in Figure 3.

[0103] The particle size measured at 14.2 nm on Pfu-Prx indicates that this protein has a decameral cyclic structure. Therefore, grafting 3C protease and PETase onto Pfu-Prx results in larger particles of 27.8 nm and 20.3 nm, respectively. This analysis demonstrates that the scaffolding for the target enzymes on the decameral Pfu-Prx ring was successfully established.

[0104] Monitoring of dissociation by isothermal titration microcalorimetry (ITC) Peroxiredoxin Tsa1 and Pfu-Prx derived from S. cerevisiae were injected into a microcalorimeter cell using a 100 μM VP-ITC stock solution (Malvern). The experiment was conducted at 25°C in 25 mM HEPES (pH 7.5) and 150 mM NaCl buffer. The results are shown in Figure 4.

[0105] The critical transition concentration (or CTC) can be determined by measuring heat release. This concentration corresponds to the concentration at which 50% of the decamer in the solution dissociates. A value of 2.8 μM was determined for Tsa1 (gray). In contrast, a value could not be determined for Pfu-Prx (black).

[0106] These results confirm that Pfu-Prx maintains its decameric cyclic structure even at very low concentrations (below the μmolar threshold). This parameter is advantageous for scaffolding function. Therefore, Pfu-Prx is a very suitable tool for the present invention.

[0107] Monitoring of thermal denaturation using differential scanning microcalorimetry (DSC) Pfu-Prx was injected into a differential scanning microcalorimeter (DSC) cell at a pressure of 2 atm using a stock solution at a concentration of 0.5 mg / ml. The experiment was conducted in 25 mM HEPES (pH 7.5) and 150 mM NaCl buffer. The results are shown in Figure 5.

[0108] The recorded thermograms showed two specific semi-denaturation temperatures (or Tm), which can be interpreted as i) the temperature at which the decamer dissociates into a dimer (93.4°C) and ii) the temperature at which the monomer denatures (107.8°C). These extremely high temperatures indicate the very high stability of the decameric structure of Pfu-Prx.

[0109] High-temperature stability is advantageous for industrial applications. Furthermore, this structural stability suggests long-term integrity of the scaffolding.

[0110] Proteolytic activity of scaffolded PreScission 3C protease The proteolytic activity of PreScission 3C, using Pfu-Prx as a scaffold, was evaluated using the 6His-SPAG1-TPR3 substrate. The latter has a specific cleavage site for the aforementioned protease. A 1 mg / mL substrate and a 2 μM scaffold protease were contacted in 1 mL of 25 mM HEPES (pH 7.5) and 150 mM NaCl buffer at 10°C for 60 minutes. Analysis was performed by electrophoresis on a denatured gel and staining with Coomassie blue. The results are shown in Figure 6.

[0111] The increased appearance of specific cleavage products over time indicates that the protease, scaffolded on Pfu-Prx, possesses proteolytic activity. The absence of spontaneous degradation of the 6His-SPAG1-TPR3 substrate in the absence of the protease was confirmed after 60 minutes at 10°C.

[0112] Decomposition test of polyethylene terephthalate (PET) using scaffolded PETase. Approximately 0.5cm 2 A PET film coupon (GoodFellow, 0.25 mm thick, transparent, amorphous) was treated with 30% ethanol for 1 hour at ambient temperature, and then dried in the open air. 100 mg of the coupon was placed in a 25 mL Erlenmeyer flask containing 5 mL of 100 mM NaPi (pH 8.00) buffer and 1 μM PETase using Pfu-Prx as a scaffold, while stirring at 50°C. The reaction was monitored for 8 days by measuring the pH of the reaction medium. A control experiment without the enzyme was performed under the same conditions. The results are shown in Figure 7.

[0113] The decrease in pH observed in the reaction medium containing the Pfu-Prx-scaffolded PETase is due to the release of terephthalic acid obtained from the degradation of PET. In the absence of the enzyme, the pH remains stable. These results indicate that the scaffolded enzyme possesses hydrolytic activity against PET.

Claims

1. 1. Use of a multimeric protein from the peroxiredoxin family as a scaffold protein, characterized in that one or more proteins of interest are linked to one or two N- and C-termini of one or more monomers of said peroxiredoxin, said peroxiredoxin having no redox activity.

2. 2. The use according to claim 1, characterized in that the one or more proteins of interest are enzymes and the scaffold protein is used to enhance the catalytic activity of the enzymes.

3. The use according to claim 1, characterized in that the proteins of interest are proteins of the same metabolic pathway and the scaffold protein is used to enhance synthesis efficiency.

4. 4. Use according to any one of claims 1 to 3, characterized in that the peroxiredoxin is the peroxiredoxin TSA1 from Saccharomyces cerevisiae according to SEQ ID NO: 1 or the peroxiredoxin from Pyrococcus furiosus according to SEQ ID NO:

16.

5. 5. The use according to claim 4, characterized in that the peroxiredoxin TSA1 contains a mutated cysteine ​​at position 48 and / or at position 171 of the sequence of SEQ ID NO:

1.

6. The use described in claim 4, characterized in that the peroxiredoxin TSA1 has the sequence of SEQ ID NO:

29.

7. 5. Use according to claim 4, characterized in that the peroxiredoxin from Pyrococcus furiosus contains a mutated cysteine ​​at position 46 and / or at position 211 of the sequence SEQ ID NO:

16.

8. The use described in claim 4, characterized in that the peroxiredoxin derived from Pyrococcus furiosus comprises the sequence of SEQ ID NO:

52.

9. The use according to any one of claims 1 to 8, characterized in that the protein of interest is fused to one or both of the N- and C-termini of the peroxiredoxin monomer via a linking sequence.

10. The use according to any one of claims 1 to 8, characterized in that the peroxiredoxin monomer and the protein of interest are linked via an adaptor / peptide ligand pair.

11. A multimeric protein derived from the peroxiredoxin family, comprising at least one protein of interest linked to one or two N- and C-termini of one or more peroxiredoxin monomers that do not have redox activity, wherein the peroxiredoxin monomer and the protein of interest are linked via an adaptor / peptide ligand pair.

12. A multimeric protein from the peroxiredoxin family, comprising at least two different proteins of interest linked to one or two N- and C-termini of one or more peroxiredoxin monomers that do not have redox activity.

13. 13. The multimeric protein according to claim 11 or 12, characterized in that the peroxiredoxin is the peroxiredoxin TSA1 from Saccharomyces cerevisiae according to SEQ ID NO: 1 or the peroxiredoxin from Pyrococcus furiosus according to SEQ ID NO:

16.

14. 14. The multimeric protein according to claim 13, characterized in that the peroxiredoxin TSA1 contains a mutated cysteine ​​at position 48 and / or at position 171 of SEQ ID NO:

1.

15. 14. The multimeric protein according to claim 13, characterized in that the peroxiredoxin from Pyrococcus furiosus contains a mutated cysteine ​​at position 46 and / or at position 211 of SEQ ID NO:

16.

16. The multimeric protein according to any one of claims 12 to 15, characterized in that the peroxiredoxin monomer and the target protein are linked via an adaptor / peptide ligand pair.

17. One or more nucleotide constructs encoding the multimeric protein according to any one of claims 12 to 16.

18. One or more nucleotide constructs encoding a multimeric protein according to any one of claims 12 to 16, comprising the sequence of SEQ ID NO: 51 encoding peroxiredoxin TSA1 mutated at positions 48 and 171 by serine and alanine, respectively, or the sequence of SEQ ID NO: 53 encoding peroxiredoxin derived from Pyrococcus furiosus mutated at positions 46 and 211 by serine and alanine, respectively.

19. An expression vector comprising the nucleotide construct of claim 18.

20. A host cell comprising a multimeric protein according to any one of claims 12 to 16, one or more nucleotide constructs according to claims 17 or 18, or an expression vector according to claim 19.

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