METHOD FOR PURIFYING A PROTEIN OF INTEREST AND MEANS FOR ITS IMPLEMENTATION

Abstract

The invention relates to a method for purifying a protein of interest, comprising the preparation of a fusion protein in which this protein of interest is fused to a protein tag comprising the Mmi1 protein of a microorganism of the genus Schizosaccharomyces or a fragment thereof, bringing this fusion protein into contact with a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif, so as to allow the affinity binding of the protein tag with this ribonucleic acid molecule, and recovering the protein of interest.

Description

Title of the invention: METHOD FOR PURIFYING A PROTEIN OF INTEREST AND MEANS FOR ITS IMPLEMENTATION ARTWORK

[0001] The present invention falls within the field of affinity purification of proteins of interest.

[0002] More particularly, the present invention relates to a method for purifying a protein of interest, as well as a recombinant fusion protein obtained during the implementation of this method. The invention also relates to a method for preparing such a fusion protein. Other objects of the invention are a solid support and a kit for implementing a purification method according to the invention.

[0003] The production in large quantities, and at low cost, of proteins of interest is proving to be of increasing interest in many sectors of the biotechnology industry.

[0004] Currently, proteins are mainly produced by culturing cell lines specially designed to express them from the genes coding for these proteins, which are integrated into these cell lines. While such biological production methods make it possible to produce the proteins of interest efficiently and in large quantities, the latter are however obtained within complex mixtures, containing in particular the elements necessary for the culture of the cell lines, as well as the other cellular components of the cells. It is therefore necessary to isolate the proteins of interest produced from these complex mixtures, in order to recover them in a form that is sufficiently pure for the intended applications, this having to be achieved without compromising the biological activity necessary for these applications.This objective is particularly crucial when the proteins of interest are, for example, hormones, antibiotic peptides, enzyme regulators, etc., or any other protein intended for therapeutic application.

[0005] Numerous methods have been proposed by the prior art for the purification of proteins of interest produced by biological processes.

[0006] Many of these methods consist of expressing the protein of interest in the form of a fusion protein in which it is associated, by genetic engineering techniques, with a protein tag having a particular affinity for a partner. It is then possible to purify the protein of interest by affinity chromatography, the partner of the protein tag being grafted onto the chromatography support.

[0007] Among such protein tags proposed by the prior art, mention may in particular be made of the histidine tag, exhibiting a high affinity for cations. cobalt and nickel, the Glutathione S-transferase tag, with high affinity for glutathione, and the maltose-binding protein (MBP), with high affinity for maltose.

[0008] As another example, document WO 2017 / 194888 describes a method for purifying proteins of interest by affinity based on the lectin activity of the CRD domain of a galectin.

[0009] However, none of the methods proposed by the prior art makes it possible to produce and purify a protein of interest at low cost, quickly, and with a high yield and high specificity leading to obtaining the protein of interest with a high purity level. The present invention aims to propose such a method.

[0010] To this end, the present invention takes advantage of the strong capacity of a particular domain of the Mmil protein of species of the genus Schizosaccharomyces to bind to a ribonucleic acid molecule (RNA) of particular sequence, with high specificity.

[0011] The Mmil protein (for Meiotic mRNA interception protein 1) plays an important role in cells in a particular post-transcriptional event, the selective elimination of meiosis-specific messenger RNAs. It has been described in the literature that this protein binds with high binding specificity to RNA, more particularly to a sequence containing repeats of the hexanucleotide UNAAAC (E. Hiriart et al., The Embo Journal, 2012, 31(10), 2296-2308; Wu et al., Biochemical and Biophysical Research Communications, 2017, 491, 310-316). This affinity has been more specifically attributed to the domain named YTH (for English YT521-B homology) of Mmil from Schizosaccharomyces, located in the C-terminal region of the protein (Stowell et al., J. Biol. Chem., 2018, 293(24), 9210-9222).

[0012] Quite surprisingly, it has been discovered by the present inventors that not only does the fusion of an Mmil protein of the species of the genus Schizosaccharomyces, or of one of its fragments comprising at least its YTH domain enlarged on the N-terminal side and on the C-terminal side (this enlarged YTH domain, consisting of the 173 C-terminal amino acids of the Mmil protein, being designated in the present description, for convenience, by the abbreviation "YTH+"), to a protein of interest, does not impact the capacity of this Mmil protein or of this fragment to bind specifically to the UNAAAC motif RNA sequence mentioned above, and this whether the fusion is carried out at the N-terminal or at the C-terminal of the Mmil protein or of its fragment;but that, in addition, bringing together a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif with a cell culture extract containing a fusion protein “protein of interest / Mmil protein or fragment of the latter containing the YTH+ domain”, does not cause massive degradation of this RNA molecule in this extract; cell culture. Thus, while a person skilled in the art would never have considered implementing, in a method for purifying a protein of interest produced by a biological method, a step of bringing together the medium containing the protein to be purified with an RNA molecule as a partner for affinity binding with the tag fused to the protein of interest, it has been discovered by the present inventors that not only is such a method entirely feasible for the particular case of the pair "Mmil from Schizosaccharomyces or fragment thereof containing the YTH+ domain / RNA molecule with UNAAAC sequence", but that it also proves to be highly efficient, since it makes it possible to purify the protein of interest with high specificity and high yield, in a short time, without altering the functions of the protein of interest.

[0013] Thus, according to a first aspect, the present invention provides a method for purifying a protein of interest, which comprises: - the preparation of a fusion protein comprising the protein of interest fused to a protein tag, said protein tag comprising at least, or consisting of: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of this Mmil protein comprising at least the 173 C-terminal amino acids (the domain of the Mmil protein formed by these 173 C-terminal amino acids being designated in the present description by the abbreviation YTH+), or a protein with an amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif with the nucleotide sequence UNAAAC, - bringing this fusion protein into contact with a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif, so as to allow the affinity binding of the protein tag with this ribonucleic acid molecule, more precisely with its UNAAAC nucleotide sequence motif, this ribonucleic acid molecule being grafted onto a solid support or coupled to a capture ligand, - where appropriate, when said ribonucleic acid molecule is coupled to a capture ligand, bringing this ribonucleic acid molecule, to which the protein of interest is linked via the protein tag, into contact with an affinity partner of the capture ligand grafted onto a solid support, - optionally, the isolation of the solid support from the medium in which it is contained, for example a cell culture medium in which the cells expressing the fusion protein have been cultured, and / or a cell lysis medium for such cells, - and the separation of the protein of interest, alone or within the fusion protein which contains it, and the solid support, to recover the protein of interest.

[0014] In the present description, the term capture ligand means a molecule which is on the one hand capable of being covalently coupled with a ribonucleic acid molecule, and on the other hand capable of binding with high affinity and specificity to an affinity partner, for example a protein receptor, which can be fixed to a solid support. As an example of such a capture ligand that can be used according to the invention, mention may be made of biotin, the affinity partners of which are avidin and streptavidin.

[0015] The method according to the invention advantageously makes it possible, by itself, to produce the protein of interest, in the form of a recombinant fusion protein, and to separate it from the production medium by taking advantage of the strong, highly specific binding capacity of the protein tag comprising the YTH+ domain of a Mmil protein of a species of the genus Schizosaccharomyces with the RNA sequence UNAAAC, to obtain the protein of interest quickly with a high degree of purity and a high yield. These steps can advantageously be carried out easily and quickly, and at low cost.

[0016] Conventionally, in the nucleotide sequence UNAAAC, U denotes uracil, A denotes adenine, C denotes cytosine and N denotes any base among adenine, cytosine, guanine and uracil. This nucleotide sequence is represented here, like all the other nucleotide sequences described, in a conventional manner, that is to say in the direction from the 5' end to the 3' end (the amino acid sequences being represented, in a conventional manner, in the reading direction from the N-terminus to the C-terminus).

[0017] The ribonucleic acid molecule containing the UNAAAC motif may comprise a single copy of this motif, or one or more repetitions of this motif. It may further comprise, 5' or 3' of this UNAAAC motif or of this series of UNAAAC motifs, one or more additional ribonucleic acids, for example 2 to 10, in particular 2 to 6, additional ribonucleic acids.

[0018] The grafting / coupling of the ribonucleic acid molecule to the solid support / capture ligand can be carried out by any conventional method in itself. This grafting / coupling is preferably carried out by covalent bonding, preferably at the 5' end or at the 3' end of the ribonucleic acid molecule.

[0019] In the present description, the term protein of interest means any protein, peptide or polypeptide, in native or recombinant form, of interest for a targeted application, in particular for an application comprising administration to a mammal, in particular a human.

[0020] The method according to the invention can for example advantageously be used for the purification of endogenous complexes in the field of fundamental research, of hormones, of antibiotic peptides or even of enzyme regulators in the field of applied research, such a list not being in any way limiting of the invention.

[0021] The protein tag used in the method according to the invention may comprise the entire Mmil protein of a microorganism of a species of the genus Schizosac-charomyces or one of its fragments containing at least the YTH+ domain (i.e. the 173 C-terminal amino acids).

[0022] The Mmil protein is preferably derived from a species selected from Schizosac-charomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus.

[0023] It is within the competence of a person skilled in the art to identify, for a given species of the genus Schizosaccharomyces, the amino acid sequence of the Mmil protein, as well as, where appropriate, the sequence of the gene coding for this protein. Such data are in particular accessible in the databases of protein sequences and nucleotide sequences. For example, in the GenBank database, the amino acid sequences of the Mmil protein are accessible, for the species Schizosaccharomyces pombe, under the accession number NP_587783.2 (SEQ ID No: 1 - the gene coding for this protein of this species has the sequence SEQ ID No: 2), for the species Schizosaccharomyces japonicus, under the accession number XP_002173827.2 (SEQ ID No: 3 - the gene coding for this protein of this species has the sequence SEQ ID No: 4), for the species Schizosaccharomyces octosporus, under the accession number XP_013019124.1 (SEQ ID No: 5 - the gene coding for this protein of this species has the sequence SEQ ID No: 6), and for the species Schizosaccharomyces cryophilus, under accession number XP_013025346.1 (SEQ ID No: 7 - the gene coding for this protein of this species has the sequence SEQ ID No: 8).

[0024] When the microorganism of the genus Schizosaccharomyces belongs to the species Schizosaccharomyces pombe, the YTH+ domain of the Mmil protein extends from the residue at position 316 (leucine residue) to the residue at position 488 (arginine residue, in the C-terminal position in the protein sequence). The YTH+ domain then has the amino acid sequence SEQ ID No: 9.

[0025] When the microorganism of the genus Schizosaccharomyces belongs to one of the species Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus, the fragment of the Mmil protein preferably contains the 174 C-terminal amino acids of the protein. The fragment of the Mmil protein used according to the invention thus preferably contains, or consists of: - for the species Schizosaccharomyces japonicus, the domain of the Mmil protein extends from the residue at position 306 (leucine residue) to the residue at position 479 (arginine residue, in the C-terminal position in the protein sequence). The domain then has for amino acid sequence the sequence SEQ ID No: 10; - for the species Schizosaccharomyces octosporus, the domain of the Mmil protein extending from the residue at position 307 (leucine residue) to the residue at position 480 (arginine residue, in the C-terminal position in the protein sequence). The domain then has the amino acid sequence SEQ ID No: 11; - for the species Schizosaccharomyces cryophilus, the domain of the Mmil protein extending from the residue at position 306 (leucine residue) to the residue at position 479 (arginine residue, in the C-terminal position in the protein sequence). The domain then has the amino acid sequence SEQ ID No: 12.

[0026] Thus, in particular embodiments of the invention, the fragment of the Mmil protein comprising at least the 173 C-terminal amino acids has an amino acid sequence comprising, or consisting of, the amino acid sequence SEQ ID No: 9 (corresponding to Schizosaccharomyces pombe), SEQ ID No: 10 (corresponding to Schizosaccharomyces japonicus), SEQ ID No: 11 (corresponding to Schizosaccharomyces octosporus) or SEQ ID No: 12 (corresponding to Schizosaccharomyces cryophilus): - SEQ ID No: 9: LNFSRASEHRNEKGERISMINPRVVLDENGISHRSRY-FIMLCDNETAIAHAKKTSIWAVKKDSSKRISDAYKKASVYFIFVAQQTYNALG YAQVVSDLNSTELPFWSDSSHAGGVRIKWIKTCNLFSAEISEIVSHMDHGSEAR DGMEMMYDEGSRLCTLINYAIMKRIGRDR - SEQ ID No: 10: LDFEHAHEYRNEKGERVSMINPRVILDENGISHRSRY-FIMLVDNETAISHAKRSSVWSLKQNYANAISEAYKQEANVYFIFINAKSYNAL GYAQLTSDVGTVAKPFWADTTYTNGVNVKWIKTCNLYSNEISEIVSRMDHGA RARDGQEMMYDEGSRLCTLINSAIMKRIGRDR - SEQ ID No: 11: LNFARASEYRNDKGERISMINPRVILDENGISSRSRY-FIMLCDNETAISHAKKTSIWAVKHDAASRVSDAYKNASIYFIFIAKPTNNALGY AQVVSDLNSAELPFWADNATYAGGVRVKWIKTCNLFSAEISDIVGRMNHGAT ARDGMEMMYDEGCRLCILVNSAIMKRIGRDR - SEQ ID No: 12: LNFARASEYRNDKGERISMINPRVILDESGISHRSRY-FIMLCDNETAIAHAKKTSIWAVKHEAASRVSDAYKKASIYFIFIAKPTNNALGY AQVVSDLNSAELPFWADNATYAGGVRVKWIKTCNLFSAEISDIVGRMNHGMT AKDGMEMMYDEGCRLCILVNSAIMKRIGRDR

[0027] The protein tag used according to the invention may also comprise, or consist of, the Mmil protein or a fragment of the Mmil protein containing at least the YTH+ domain of the protein, or a protein with an amino acid sequence having at least 90%, preferably at least 95%, preferentially at least 98% and more preferably at least 99%, identity with the amino acid sequence of the Mmil protein or said fragment of said Mmil protein, and capable of binding to a ribonucleic acid motif of nucleotide sequence UNAAAC, preferably with a specificity at least as good as the YTH+ domain of the protein.

[0028] For any protein of given amino acid sequence having at least 90%, preferably at least 95%, preferentially at least 98% and more preferably at least 99%, identity with the amino acid sequence of the Mmil protein or said fragment of the Mmil protein, it is within the skill of the person skilled in the art to evaluate its capacity to bind to the RNA motif of sequence UNAAAC, by binding tests which are conventional in themselves, for example by gel retardation tests after electrophoresis or fluorescence spectroscopy, circular dichroism or plasmon resonance. The binding specificity can be evaluated by these same methods, by comparison with RNA molecules of sequence close to the UNAAAC sequence (for example the sequence CNAAAC or GNAAAC) and to compare the results obtained with those obtained with said Mmil protein or said fragment of the Mmil protein.

[0029] The protein with an amino acid sequence having at least 90%, preferably at least 95%, preferentially at least 98% and more preferably at least 99%, identity with the amino acid sequence of the Mmil protein or said fragment of the Mmil protein may have, with respect to the sequence of the Mmil protein or said fragment of the Mmil protein, which constitutes the reference sequence, insertions, deletions and / or substitutions. In the case of a substitution, this is preferably carried out by an amino acid of the same family as the original amino acid, for example by substitution of a basic residue such as arginine by another basic residue such as a lysine residue, of an acidic residue such as aspartate by another acidic residue such as glutamate, of a polar residue such as serine by another polar residue such as threonine, of an aliphatic residue such as leucine by another aliphatic residue such as isoleucine, etc.

[0030] The percentage identity between two amino acid sequences is here determined in a conventional manner in itself, by comparing the two optimally aligned sequences, through a comparison window, the part of the amino acid sequence to be compared located in the comparison window being able to comprise additions or deletions with respect to the reference sequence so as to obtain an optimal alignment between the two sequences. The percentage identity is then calculated by determining the number of positions for which an amino acid residue is identical in the two sequences compared, then dividing this number of positions by the total number of positions in the comparison window, the number obtained being multiplied by one hundred to obtain the percentage identity between the two sequences.

[0031] The method according to the invention can furthermore meet one or more of the characteristics characteristics described below, implemented in isolation or in each of their technically effective combinations.

[0032] Within the fusion protein, the protein tag may be fused to the N-terminus or the C-terminus of the protein of interest.

[0033] Optionally, a spacer may be integrated between the protein of interest and the protein tag.

[0034] In particular embodiments of the invention, an enzymatic cleavage site is inserted between the protein of interest and the protein tag. Any conventional cleavage site in itself falls within the scope of the invention, in particular protease cleavage sites such as the cleavage site of the TEV protease (tobacco etch virus protease).

[0035] In such embodiments, the method according to the invention preferably comprises a step of cleaving the fusion protein, by a suitable enzyme, at this enzymatic cleavage site, so as to separate the protein of interest from the protein tag.

[0036] Such a step of cleaving the fusion protein is however only optional, and is in particular only necessary when the fusion of the protein tag to the protein of interest modifies the activity of the latter useful for the intended application of the protein of interest. When the fusion of the protein tag to the protein of interest does not modify the activity of the latter, and also does not induce any harmful side effect in the context of the intended application, then a step of separating the protein of interest and the protein tag is not at all necessary.In particular, Mmil proteins of the genus Schizosaccharomyces interact with RNA in a completely different way from mammalian YTH domain proteins, so that the fusion protein prepared and purified according to the invention can advantageously be administered, as such, to a mammal, in particular to a human, without the protein tag disrupting normal biological processes.

[0037] In a completely advantageous manner, the presence of the protein tag according to the invention in fusion with the protein of interest does not cause any undesirable effect in the cells of higher eukaryotes. This protein tag does not present any toxicity for these cells, and does not cause any modification of the localization of the protein of interest there.

[0038] The step of preparing the fusion protein can be carried out in any conventional manner, in particular biologically, by implementing conventional genetic engineering techniques per se.

[0039] In particular, the fusion protein may be prepared by transfecting a suitable host organism with a nucleic acid molecule encoding the fusion protein, or transforming a suitable host organism with a vector expression in which the hybrid gene encoding the fusion protein is operably linked to a DNA sequence controlling its expression; and culturing this host organism under conditions allowing the expression of the fusion protein, such conditions being conventional in themselves and well known to those skilled in the art.

[0040] Host organisms that may be used for this purpose include, but are not limited to, gram-positive and gram-negative bacteria such as strains of Escherichia coli or Bacillus subtilis, yeasts such as strains of Saccharomyces cerevisiae, and higher eukaryotic organisms, including mammalian cell lines.

[0041] The hybrid gene encoding the fusion protein can be prepared by conventional recombinant DNA methods or by gene synthesis methods also conventional in themselves. It can be incorporated into any conventional protein expression vector.

[0042] At the end of this preparation step of the method according to the invention, the fusion protein produced is separated from the host organism and the culture medium by placing the target RNA molecule, grafted onto a solid support or covalently coupled to a capture ligand, in the medium containing it, where appropriate after cell lysis (such a cell lysis step not being necessary when the fusion protein to be separated from the medium is of the extracellular addressing type).

[0043] In particular embodiments of the invention, this ribonucleic acid molecule contains one or more repetitions of the sequence motif UNAAAC. These different repetitions can be contiguous or spaced from each other by a spacer sequence.

[0044] The ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif may comprise, within this motif and / or in any other position: - at least one chemically modified nucleotide, in particular a 2'-O-methylated nucleotide, such modification being carried out in a conventional manner in itself, in particular at the level of the ribose motif; - and / or at least one locked nucleic acid, called LNA (for the English “locked nucleic acid”), that is to say a nucleic acid analogue containing a methylene bridge between the hydroxyl in position 2 and the atom in position 4 of the sugar; - and / or at least one phosphorothiate internucleotide bond.

[0045] The step of bringing the fusion protein into contact with the ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif is preferably carried out for a period of between 5 minutes and 1 hour, preferably between 10 minutes and 30 minutes.

[0046] The solid support onto which the affinity partner of the capture ligand or the ribonucleic acid molecule is grafted may be of any conventional type in itself, in particular for carrying out separation methods by immunoprecipitation. It may, for example, consist of agarose or polystyrene beads, for example with magnetic properties, which may in particular be isolated from the medium containing them by magnets.

[0047] In particular embodiments of the invention, the solid support is a chromatography support, conventional in itself, for example a crosslinked polymer based on polysaccharide, such as dextran or agarose, or polyacrylamide, in particular in the form of porous beads, or polystyrene.

[0048] For the implementation of the method according to the invention, the chromatography support is then preferably contained in a column (or tube), into which the medium containing the fusion protein is introduced, where appropriate linked to the ribonucleic acid molecule when the method according to the invention uses a capture ligand coupled to the latter, for the attachment of the fusion protein to the chromatography support according to the principle of affinity chromatography, this affinity being either between the capture ligand and its affinity partner grafted onto the support, or between the fusion protein, more precisely the protein tag, and the ribonucleic acid molecule grafted onto the support.

[0049] Such a chromatography column can be operated in batch or continuous mode.

[0050] Preferably, prior to its being brought into contact with the fusion protein, the chromatography support is equilibrated, in a conventional manner in itself and according to the manufacturer's recommendations, in particular by an aqueous equilibration buffer. This equilibration buffer may contain a denaturing agent or a detergent such as guanidinium chloride, urea or Triton® X-100.

[0051] The separation of the fusion protein and the solid support can be carried out in different ways, all comprising a step of elution of the protein of interest (alone or in the form of fusion protein with the protein tag). This elution can be carried out at constant pH or with linearly or discontinuously decreasing pH gradients. The optimal elution conditions are easily determined by a person skilled in the art, by routine experiments, for each given protein of interest.

[0052] The elution buffer may contain a denaturing agent or detergent such as guanidinium chloride, urea or Triton® X-100. The addition of such a denaturing agent or detergent facilitates purification, even when the protein of interest is poorly soluble in aqueous solution.

[0053] In particular embodiments of the invention in which a site of enzymatic cleavage is inserted between the protein of interest and the protein tag, separation of the protein of interest from the solid support can be achieved by cleavage at the enzymatic cleavage site. The protein of interest can then be recovered by elution, as described above.

[0054] In variants of the invention, the fusion protein can be separated from the solid support by chemical elution, in particular by means of a low pH glycine-based solution, then the protein of interest can optionally be dissociated from the protein tag by cleavage at the enzymatic cleavage site which separates them.

[0055] In particular embodiments of the invention, a cleavage site is inserted between the ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif and the solid support or the capture ligand. This cleavage site may be of the type cleavable by a specific RNAse enzyme. In such a configuration, the separation of the protein of interest and the chromatography support may be carried out by cleavage at this cleavage site. The fusion protein may then be recovered by elution, as indicated above.

[0056] The separation of the fusion protein and the solid support can otherwise be achieved by chemical cleavage within the immobilized RNA molecule, directly or indirectly, via the "capture ligand / affinity partner" pair, on the solid support.

[0057] In particular embodiments of the invention, the protein tag contains, in addition to the fragment of the Mmil protein containing the 173 or 174 C-terminal amino acids, at least one domain chosen from the Schizosaccharomyces Mmil domains of the following amino acid sequence: - RSVWXaaiXaa2Xaa3Xaa4Xaa5Xaa6P (SEQ ID No: 13) (Schizosaccharomyces Mmil domain 2), where Xaai represents a threonine, serine or alanine residue, Xaa2 represents a threonine, arginine, lysine or serine residue, Xaa3 represents a histidine or arginine residue, Xaa4 represents a threonine or proline residue, Xaa5 represents a glycine, alanine or arginine residue and Xaa6 represents a glutamic acid or aspartic acid residue, this domain being referred to in the present description as "domain 2"; - FXaa7SPLKRXaa8APXaa9SXaaioXaaiiXaai2Xaai3Xaai4Xaai5R (SEQ ID No: 14) (Schizosaccharomyces Mmil domain 3), where Xaa7 represents a serine or threonine residue, Xaa8 represents a proline or glycine residue, Xaa9 represents a glutamic acid or aspartic acid residue, Xaaio represents a histidine, arginine or lysine residue, Xaan represents an aspartic acid or glutamic acid residue, Xaa[2 represents an alanine or tyrosine residue, Xaa[3 is zero or represents a proline residue, Xaa[4 represents an isoleucine or methionine residue and Xaai5 represents a glycine or aspartic acid residue, this domain being designated in the present description as “domain 3”; - YDFXaa16RHCTDYGHSYXaa17WPYFRSXaa18RREXaa19Xaa20Xaa21Y (SEQ ID No: 15) (Schizosaccharomyces Mmil domain 4), where Xaa[6 represents a serine, threonine or tyrosine residue, Xaan represents a glutamic acid or aspartic acid residue, Xaa[8 represents a leucine or valine residue, Xaai9 is null or represents a serine residue, Xaa20 represents a leucine or methionine residue and Xaa2i represents an arginine, leucine or methionine residue, this domain being referred to in the present description as “domain 4”; - QPPXaa22KRRTLXaa23Xaa24P (SEQ ID No: 16) (Schizosac-charomyces Mmil domain 5), where Xaa22 represents a proline, serine or leucine residue, Xaa23 represents a serine or leucine residue and Xaa24 represents a proline or serine residue, this domain being referred to in the present description as “domain 5”; - Xaa25AXaa26Xaa27SPXaa28Xaa29Xaa3oXaa3iPXaa32Xaa33H (SEQ ID No: 17) (Schizosaccharomyces Mmil domain 6), where Xaa25 represents an arginine or aspartic acid residue, Xaa26 represents a serine or glycine residue, Xaa27 represents a histidine or aspartic acid residue, Xaa28 represents a serine, glycine or leucine residue, Xaa 29 represents a leucine or phenylalanine residue, Xaa30 represents a leucine, isoleucine or serine residue, Xaa3[ represents a glutamic acid or aspartic acid residue, Xaa32 represents a tyrosine or threonine residue and Xaa33 represents an alanine or threonine residue, this domain being designated in the present description as “domain 6”; - RXaa34EKPKXaa35RAXaa36TPPP (SEQ ID No: 18) (Schizosaccharomyces Mmil domain 7), where Xaa34 represents a lysine or arginine residue, Xaa35 represents an alanine, proline or threonine residue and Xaa36 represents a serine or proline residue, this domain being referred to in the present description as "domain 7".

[0058] The protein tag may contain two or more of the above domains 2 to 7, including all of these domains, with any combination of two or more of these domains being within the scope of the invention.

[0059] Preferably, in the amino acid sequence of each of domains 2 to 7 above, the variable amino acids are chosen so that the amino acid sequence of each of the domains corresponds to the amino acid sequence of a domain of the Mmil protein of a microorganism of the genus Schizosaccharomyces, preferably the same as that from which said fragment containing the YTH+ domain of the protein originates. The different domains, as well as the YTH+ domain, contained in the protein tag according to the invention are then preferably positioned relative to each other according to their normal positioning in the native protein, these domains then being contiguous or separated by the native intercalary sequences of the protein, or by sequences obtained by substitution, addition or deletion by compared to these native intercalary sequences.

[0060] In particular embodiments of the invention, the protein tag comprises the YTH+ domain of the Mmil protein of Schizosaccharomyces pombe and at least one domain of this protein chosen from the following domains, or several of these domains, or even all of them, according to all possible combinations: - domain 2: extending from the amino acid in position 40 (arginine) to the amino acid in position 50 (proline) of the Mmil protein, of sequence: RSVWTTHTGEP (SEQ ID No: 19), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 3: extending from the amino acid in position 64 (phenylalanine) to the amino acid in position 82 (arginine) of the Mmil protein, of sequence: FSSPLKRPAPE-SHDAPIGR (SEQ ID No: 20), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 4: extending from the amino acid in position 97 (tyrosine) to the amino acid in position 125 (tyrosine) of the Mmil protein, of sequence: YDFSRHCTDYGHSYEW-PYFRSLRRESMLY (SEQ ID No: 21), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 5: extending from the amino acid in position 169 (glutamine) to the amino acid in position 180 (proline) of the Mmil protein, of sequence: QPPPKRRTLSPP (SEQ ID No: 22), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 6: extending from the amino acid in position 258 (arginine) to the amino acid in position 272 (histidine) of the Mmil protein, of sequence: RASHSPSLLEPYAH (SEQ ID No: 23), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 7: extending from the amino acid in position 302 (arginine) to the amino acid in position 315 (proline) of the Mmil protein, of sequence: RKEKPKARASTPPP (SEQ ID No: 24), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence.

[0061] As indicated above, these different domains, as well as the YTH+ domain, contained in the protein tag, are preferably positioned relative to each other according to their normal positioning in the native protein, these domains then being contiguous or separated by the native intercalary sequences of the protein, or by sequences obtained by substitution, addition or deletion relative to these native intercalary sequences.

[0062] In particular embodiments of the invention, the protein tag comprises, or consists of, the Mmil protein of Schizosaccharomyces pombe deleted by the 30 amino acids in the N-terminal position. The protein tag then comprises, or consists of, the amino acid sequence of sequence SEQ ID No: 25.

[0063] In particular embodiments of the invention, the protein tag comprises the YTH+ domain of the Mmil protein of Schizosaccharomyces japonicus and at least one domain of this protein chosen from the following domains, or several of these domains, or even all of them, according to all possible combinations: - domain 2: extending from the amino acid in position 38 (arginine) to the amino acid in position 48 (proline) of the Mmil protein, of sequence: RSVWAKHPNDP (SEQ ID No: 26), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 3: extending from the amino acid in position 61 (phenylalanine) to the amino acid in position 78 (arginine) of the Mmil protein, of sequence: FSSPLKR-GAPDSKEYMDR (SEQ ID No: 27), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 4: extending from the amino acid in position 93 (tyrosine) to the amino acid in position 120 (tyrosine) of the Mmil protein, of sequence: YDFYRHCTDYGH-SYDWPYFRSLRRELAY (SEQ ID No: 28), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 5: extending from the amino acid in position 166 (glutamine) to the amino acid in position 177 (proline) of the Mmil protein, of sequence: QPPLKRRTLLSP (SEQ ID No: 29), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 6: extending from amino acid position 245 (aspartic acid) to amino acid position 258 (histidine) of the Mmil protein, sequence: DAGD- SPLFSEPTAH (SEQ ID No: 30), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferably at least 99%, identity with this amino acid sequence; - domain 7: extending from the amino acid in position 292 (arginine) to the amino acid in position 305 (proline) of the Mmil protein, of sequence: RREKPKTRAPTPPP (SEQ ID No: 31), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence.

[0064] In alternative embodiments of the invention, the protein tag comprises the YTH+ domain of the Mmil protein of Schizosaccharomyces octosporus and at least one domain of this protein chosen from the following domains, or several of these domains, or even all of them, according to all possible combinations: - domain 2: extending from the amino acid in position 39 (arginine) to the amino acid in position 49 (proline) of the Mmil protein, of sequence: RSVWSNRPAEP (SEQ ID No: 32), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 3: extending from the amino acid in position 61 (phenylalanine) to the amino acid in position 79 (arginine) of the Mmil protein, of sequence: FTSPLKRPAPD-SREAPMGR (SEQ ID No: 33), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 4: extending from the amino acid in position 94 (tyrosine) to the amino acid in position 122 (tyrosine) of the Mmil protein, of sequence: YDFTRHCTDYGHSYEW-PYFRSVRRESLMY (SEQ ID No: 34), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 5: extending from the amino acid in position 170 (glutamine) to the amino acid in position 181 (proline) of the Mmil protein, of sequence: QPPSKRRTLSPP (SEQ ID No: 35) or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 6: extending from amino acid position 253 (arginine) to amino acid position 266 (histidine) of the Mmil protein, sequence: RASHSPGLIDPYTH (SEQ ID No: 36), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferably at least 99%, identity with this amino acid sequence; - domain 7: extending from the amino acid in position 293 (arginine) to the amino acid in position 306 (proline) of the Mmil protein, of sequence: RKEKPKPRAPTPPP (SEQ ID No: 37), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence.

[0065] In still alternative embodiments of the invention, the protein tag comprises the YTH+ domain of the Mmil protein of Schizosaccharomyces cryophilus and at least one domain of this protein chosen from the following domains, or several of these domains, or even all of them, according to all the possible combinations: - domain 2: extending from the amino acid in position 39 (arginine) to the amino acid in position 49 (proline) of the Mmil protein, of sequence: RSVWSSRPAEP (SEQ ID No: 38), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 3: extending from the amino acid in position 61 (phenylalanine) to the amino acid in position 79 (proline) of the Mmil protein, of sequence: FTSPLKRPAPD-SREAPIGR (SEQ ID No: 39), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 4: extending from the amino acid in position 94 (tyrosine) to the amino acid in position 122 (tyrosine) of the Mmil protein, of sequence: YDFTRHCTDYGHSYEW-PYFRSVRRESLMY (SEQ ID No: 40), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 5: extending from the amino acid in position 169 (glutamine) to the amino acid in position 180 (proline) of the Mmil protein, of sequence: QPPSKRRTLSPP (SEQ ID No: 41) or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 6: extending from amino acid position 252 (aspartic acid) to amino acid position 265 (histidine) of the Mmil protein, of sequence: RASHSPSLIDPYAH (SEQ ID No: 42), or a protein region of amino acid sequence amino acids having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence; - domain 7: extending from the amino acid in position 292 (arginine) to the amino acid in position 305 (proline) of the Mmil protein, of sequence: RKEKPKPRAPTPPP (SEQ ID No: 43), or a protein region of amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 98%, and preferentially at least 99%, identity with this amino acid sequence.

[0066] The fusion protein according to the invention may further comprise other chemical or protein tags, conventional in themselves, one or more subcellular localization sequences, etc.

[0067] Another aspect of the present invention relates to a recombinant fusion protein, capable of being obtained at the end of the step of preparing a fusion protein of the purification method according to the invention, or at the end of the method according to the invention, in purified form, comprising a protein of interest fused to a protein tag, this protein tag comprising at least, or consisting of: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of this Mmil protein comprising at least the 173 C-terminal amino acids, or a protein with an amino acid sequence having at least 90% identity, preferably at least 95%, preferentially at least 98%, and more preferentially at least 99% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif of nucleotide sequence UNAAAC.

[0068] This fusion protein may have any characteristic or combination of characteristics described above with reference to the purification method according to the invention, and relating to the fusion protein prepared / implemented.

[0069] In particular, an enzymatic cleavage site is inserted between the protein of interest and the protein tag.

[0070] Another aspect of the invention relates to a nucleic acid molecule encoding a fusion protein according to the invention. This nucleic acid molecule, particularly suitable for implementation in the step of preparing the fusion protein of a purification method according to the invention, can in particular be obtained by any conventional genetic engineering method in itself.

[0071] The nucleic acid molecule according to the invention may for example comprise, in the reading frame, a sequence chosen from the sequences SEQ ID No: 2 and SEQ ID No: 44, corresponding to the sequences coding, respectively, for the Mmil protein of Schizosaccharomycespombe, of amino acid sequence SEQ ID No: 1, and for the YTH+ domain of the latter, of amino acid sequence SEQ ID No: 9.

[0072] The invention also relates to an expression vector comprising a nucleic acid molecule according to the invention. This expression vector, particularly suitable for implementation in the step of preparing the fusion protein of a purification method according to the invention, may be of any type known per se for implementation in genetic engineering, in particular a plasmid, a cosmid, a virus, a bacteriophage, containing the elements necessary for the transcription and translation of the sequence coding for the fusion protein according to the invention.

[0073] It comprises in particular the following elements, functionally linked: a promoter located 5' of a nucleotide sequence coding for the fusion protein according to the invention, and transcription termination signals 3' of this sequence.

[0074] The present invention also relates to a host cell comprising a fusion protein, a nucleic acid molecule and / or an expression vector according to the invention. This host cell, particularly suitable for implementation in the step of preparing the fusion protein of a purification method according to the invention, may be a prokaryotic cell, in particular a bacterial cell, in particular for the mass production of the fusion protein according to the invention, or a eukaryotic cell, this eukaryote being able to be lower or higher, for example a yeast, invertebrate or mammalian cell. In particular, cell lines expressing, in a stable, inducible or constitutive manner, or transiently, a fusion protein according to the invention fall within the scope of the invention.

[0075] The fusion protein according to the invention can be prepared by any conventional method known to those skilled in the art. It can in particular be obtained by genetic engineering or by chemical synthesis.

[0076] The invention also relates to a method for preparing a fusion protein according to the invention, this method being able to be implemented to carry out the step of preparing the fusion protein of the purification method according to the invention. This preparation method comprises the transfection of a host cell with a nucleic acid molecule according to the invention or the transformation of a host cell with an expression vector according to the invention; and the culture of this host cell under conditions allowing the expression of the targeted fusion protein.

[0077] Another subject of the invention is a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif, coupled to a capture ligand, for example to a biotin molecule.

[0078] This ribonucleic acid molecule may have any characteristic or combination of characteristics described above with reference to the purification method according to the invention, and relating to the ribonucleic acid molecule used.

[0079] Another aspect of the invention is a solid support for implementing a method for purifying a protein of interest according to the invention. A ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif is grafted onto this solid support.

[0080] This solid support may have any characteristic or combination of characteristics described above with reference to the purification method according to the invention, and relating to the grafted solid support used.

[0081] According to another aspect, the invention relates to a kit for implementing a method for purifying a protein of interest according to the invention. This kit contains at least two of the following constituents, for example these three constituents: - a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif grafted onto a solid support or coupled to a capture ligand; - an expression vector comprising, under the control of a promoter, a nucleic acid molecule coding for a protein tag comprising at least, or consisting of: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of this Mmil protein comprising at least the 173 C-terminal amino acids, or a protein with an amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif with the nucleotide sequence UNAAAC; and a site allowing the insertion, 5' or 3' relative to said nucleic acid molecule coding for said protein tag, of a nucleic acid molecule coding for said protein of interest so as to allow the expression of a fusion protein containing said protein of interest and said protein tag; - and / or instructions for implementing the steps of a method according to the invention.

[0082] This kit may further contain, in the case in which the ribonucleic acid molecule is coupled to a capture ligand, a solid support onto which an affinity partner of this capture ligand is grafted.

[0083] Each of these constituents can meet one or more of the characteristics described above with reference to the purification process according to the invention, and relating to this constituent.

[0084] The expression vector, allowing the cloning of the nucleic acid molecule coding for the protein of interest so as to form a chimeric nucleic acid molecule coding for the fusion protein, and the expression of the latter, can be of any type known in itself for implementation in genetic engineering, in particular a plasmid, a cosmid, a virus, a bacteriophage, etc. It contains the elements necessary for cloning into a suitable insertion site of a molecule of nucleic acid coding for the protein of interest, as well as the transcription and translation of the chimeric sequence coding for this fusion protein "protein of interest - protein tag" (or "protein tag - protein of interest") thus obtained. It comprises in particular the following elements, functionally linked: a promoter located 5' of a nucleic acid molecule coding for the protein tag according to the invention, a site for insertion of a nucleic acid molecule coding for the protein of interest in the same reading frame as the nucleic acid molecule coding for the protein tag according to the invention, and transcription termination signals 3' of these elements.

[0085] The kit may also contain a host cell capable of being transformed by an expression vector comprising, under the control of a promoter: a nucleic acid molecule coding for a protein tag comprising at least, or consisting of, the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of this Mmil protein comprising at least the 173 C-terminal amino acids, or a protein with an amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif with the nucleotide sequence UNAAAC; and a site allowing the insertion, 5' or 3' relative to this nucleic acid molecule coding for this protein tag, of a nucleic acid molecule coding for the protein of interest, this site being configured to allow the expression of a fusion protein containing the protein of interest and the protein tag.

[0086] This host cell can be either a prokaryotic cell, in particular a bacterial cell, in particular for the mass production of the fusion protein according to the invention, or a eukaryotic cell, this eukaryote being able to be lower or higher, for example a yeast, invertebrate or mammalian cell.

[0087] The characteristics and advantages of the invention will appear more clearly in the light of the following examples of implementation, provided for purely illustrative purposes and in no way limiting the invention, with the support of figures 1 to 6, in which:

[0088] [Fig-1] [Fig.l] shows a photograph of a western blot membrane, the re velation having been carried out using an anti-Mmil antibody, obtained after expression of the Mmil protein of S. pombe in E. coli, cell lysis, placing in the presence (right lane, “WT RNA”) or not (left lane, “Control”) for 15 min of the lysate diluted to 1 / 100th with an RNA molecule “WT RNA” containing a UNAAAC motif (in accordance with the invention), recovery of the proteins bound to the RNA molecule, and separation of the proteins by SDS electrophoresis.

[0089] [Fig.2] [Fig.2] shows a photograph of a western blot membrane, the re velation having been carried out by means of an anti-Mmil antibody, obtained after expression of the Mmil protein of S. pombe in E. coli, cell lysis, implementation presence of the lysate diluted to 1 / 10 with a “WT RNA” RNA molecule containing a UNAAAC motif (in accordance with the invention, middle lane) or a “mutated RNA” RNA molecule containing a CNAAAC motif (not in accordance with the invention - negative control, right lane) or absence of such presence (left lane, “Control”), recovery of the proteins bound to the RNA molecule, and separation of the proteins by SDS-PAGE electrophoresis.

[0090] [Fig.3] [Fig.3] shows a photograph of a western blot membrane, the revelation having been carried out using an anti-Mmil antibody, obtained after expression of the S. pombe Mmil protein in S. pombe, cell lysis, bringing into contact (or absence of bringing into contact: left lane, “Control”) for 1 h the lysate diluted to 1 / 10 with an anti-Mmil antibody (right lane) or an antibody not directed against Mmil (human IgG, middle lane, “Non-spec.”), recovery of the proteins bound to the antibody, and separation of the proteins by SDS-PAGE electrophoresis.

[0091] [Fig.4] [Fig.4] shows a photograph of a western blot membrane, the re velation having been carried out using an anti-Mmil antibody, obtained after expression of the Mmil protein of S. pombe in fusion with protein A (right lane, “Mmil-TAP”) or alone (middle lane, “Mmil”) in S. pombe, cell lysis, bringing into contact (or absence of bringing into contact: left lane, “Control”) for 1 h the lysate diluted to 1 / 10 with IgG grafted onto beads, recovery of the proteins bound to the IgG, and separation of the proteins by SDS-PAGE electrophoresis.

[0092] [Fig.5] [Fig.5] shows photographs of western blot membranes, the revelation having been carried out by means of an anti-GFP antibody, obtained after expression in HEK293 cells, respectively, in a / of GFP and in b / of the Mmil protein fused to GFP.

[0093] [Fig.6] [Fig.6] shows images, acquired by fluorescence microscopy, of HEK293 cells overexpressing, in a / the fusion protein of GFP and the Mmil protein, the GFP being on the C-terminal side, in b / GFP alone, and in c / the fusion protein of GFP and the Mmil protein, the GFP being on the N-terminal side.

[0094] A / Materials and methods

[0095] Al / Cells and plasmids used for the production of proteins

[0096] - Bacteria: Escherichia coli BL21 strain and plasmid pETMl 1 (the sequences DNA sequences encoding the Mmil protein were cloned into this plasmid using the NcoI and EcoRI restriction sites); - Yeasts: Schizosaccharomyces Pombe; - Human cells: HeLa and HEK293 lines, and plasmids pEGFP-C3 and pEGFP-N3 (Clontech) (the DNA sequences coding for the Mmil protein were cloned into these plasmids using the XhoI and BamHI restriction sites).

[0097] In all experiments, the Mmil protein is that of S. pombe (Mmil protein complete, of amino acid sequence SEQ ID No: 1).

[0098] A.2 / Bacterial expression

[0099] D1: Transform the cells of the BL21 strain with the plasmid pETMl 1 containing Mmil following the standard procedure used for Top 10 cells (applying the conventional bacterial transformation protocol); D2: Pick a colony and inoculate it into 50 ml of LB medium supplemented with 50 mM kanamycin and let the culture grow at 37 °C with shaking (180 rpm); D3: Transfer the inoculum into 200 ml of LB medium supplemented with 50 mM kanamycin and shake it again at 37 °C for 1 h;check the optical density of the cells - when it reaches 0.8, take an aliquot of uninduced cells (5 ml), in order to constitute the negative control of induction (uninduced cells) - lower the temperature of the incubator to 18 °C and leave the culture shaking for 15 min before inducing expression by adding IPTG - add 0.2 ml of IPTG IM in a 200 ml culture (final concentration 1 mM) and leave the culture shaking overnight; D4: centrifuge the cells in 50 ml fractions at 5000 g for 20 min at 4 °C, then freeze the pellets at -80 °C. ;

[0100] A.3 / Binding of Mmil to RNA in E. coli

[0101] Cell lysis: A pellet of E. coli BL21 cells expressing the Mmil protein is thawed on ice. The cells are then lysed by sonication in 5 ml of lysis buffer (50 mM Tris pH 7.6, 150 mM KCl, 5 mM MgCl2, 1 mM EDTA, 1% Triton X-100, 10% glycerol, 5 mM DTT, 1 mM PMSF, 1 µg / ml of LABP (Protease inhibitor cocktail (Sigma): Aprotinin ref. 10236624001, Bestatin ref. 10874515001, Leupeptin ref. 10874515001, Pepstatin ref. 11359053001). The lysate is then transferred to a new 15 ml tube and centrifuged for 10 min at 14000 g at 4 °C, then sonicated according to a alternating “10 s sonication / 10 s rest” at 85% of maximum power (Vibracell 75186 sonicator, ThermoFisher) for a total sonication time of 2 min.

[0102] Preparation of biotinylated RNAs: for each immunoprecipitation, 100 ng of the RNAs named “WT RNA” and “mutated RNA” are denatured for 2 min at 90 °C, then the denatured RNAs are incubated for 20 min at room temperature in the structuring buffer (10 mM Tris pH 7, 0.1 M KC1, 10 mM MgCl2) before being incubated with the lysate. The RNAs used were obtained from Eurogentec and have the following sequences: WT RNA: 5' Biot-GGAUCCUUAAACAGAUCU 3' (SEQ ID No: 45) (artificial sequence, substrate for Mmil binding) - presenting a UNAAAC motif in accordance with the invention, Mutated RNA: 5' Biot-GGAUCCCUAAACAGAUCU 3' (SEQ ID No: 46) (artificial sequence, negative control for Mmil binding) - negative control not showing of UNAAAC pattern but a CNAAAC pattern.

[0103] RNA binding: a 1 / 100 or 1 / 10 dilution of the cell extract obtained at the end of the cell lysis step is carried out. The biotinylated RNA (100 ng) is added to 100 μl of cell extract, then the extract is incubated with shaking for 15 to 20 min at 4 °C. Immunoprecipitation is carried out by adding 15 μl of 10 mg / ml of Dynabeads® M-280 Streptavidin (Invitrogen) and incubation for 30 min with shaking. The beads are then washed three times with 1 ml of lysis buffer with 5 min of shaking after each wash. The elution of the Mmil protein associated with the RNA is carried out by boiling for 5 min in 2X Laemmli SDS buffer. The efficiency of protein binding to RNA is analyzed by immunoblotting (western blot), with separation by SDS-PAGE electrophoresis being carried out on a 10% polyacrylamide gel for 1 h at 180 V.After transfer to nitrocellulose membrane, protein detection is performed using an anti-Mmil antibody, as described in the publication by Touat-Todeschini et al, EMBO J, 2017, 36:2626-2641. For this purpose, the nitrocellulose membrane is incubated for one hour at room temperature with this primary anti-Mmil antibody diluted 1 / 1000 in TBS 0.1% tween (TBS-T) containing 10% FCS, followed by three washes of 5 min each with TBS-T. The membrane is then incubated for 45 min with a secondary antibody coupled to HRP (DAKO, ref. P0448) diluted 1 / 5000 in TBS-T containing 1% milk, then washed again 3 times 5 min with TBS-T.

[0104] A.4 / Immunoprecipitation experiments in yeast

[0105] S. pombe cells are used in which Mmil has been overexpressed using an inducible promoter (nmtl promoter) inserted at the endogenous Mmil locus. Cells overexpressing Mmil in fusion with the TAP tag (tandem affinity purification) composed of protein A and CBP (calmodulin-binding protein) are also used, in which the TAP tag has been added to the endogenous mmil gene sequence by the conventional yeast homologous recombination polymerase chain reaction approach. These cells are grown to an optical density (OD) of 1.2 in a total volume of 50 ml of YEA culture medium.

[0106] The following steps are all carried out at 4°C.

[0107] Lysis buffer (LysBuff) (100 mM HEPES pH 7.5, 20 mM MgCl2, 10% Glycerol, 10 mM EGTA, 0.1 M EDTA, 0.4% NP-40, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 1 pg / ml LABP) is used for lysis and for washing.

[0108] Cell lysis: Resuspend the pellets in 400 µl of LysBuff lysis buffer (2x) (mix the tube gently if necessary), then add glass beads and shake for 2x30 s in a bead shaker, with a rest time of 2 min in ice between the two cycles. Pierce the bottom of the tubes with a 0.5 mm syringe and place the tubes of lysate into 5 ml round-bottom tubes (ice-chilled). Centrifuge at 4 °C for 1 min at 3000 rpm, then transfer the lysates into new Eppendorf tubes. Centrifuge for 10 min at 13000 rpm at 4 °C. The protein concentration in each sample obtained is estimated by the Bradford method, so that the same amount of total protein is used in each immunoprecipitation experiment.

[0109] TAP immunoprecipitation: Use 15 µl of IgG antibodies grafted onto Sepharose® resin beads (IgG Sepharose, Ref. 17-0969-01, GE-Healthcare) per immunoprecipitation experiment. Wash the beads 3 times with 500 µl of 2x LysBuff lysis buffer. Mix the protein samples (the same amount of protein in each sample) with 15 µl of beads prepared in the previous step. Shake gently at 4°C for 1 h.

[0110] Immunoprecipitation by anti-Mmil antibody: the proteins (the same quantity in each sample) are mixed with 2 μl of the aforementioned anti-Mmil antibody and the mixture is gently stirred for 1 h at 4°C. 20 μl of Sepharose® resin beads on which protein A is grafted (Sepharose® Protein A, Ref. 17-5280-01, GE-Healthcare) are added to the mixture, which is then stirred again for 1 h at 4°C.

[0111] Washes and elution: wash the beads 3 times with 500 µl of 2x LysBuff using the same method: wash for 5 min with gentle agitation then centrifugation for 2 min at 500 g. Elution is carried out by boiling for 5 min in 2X Laemmli SDS buffer. For Western Blot detection, the same procedure as that described above with reference to the experiment on binding of Mmil to RNA in E. coli is used. TAP detection is carried out with an anti-TAP antibody.

[0112] A.5 / Transfection of human cells

[0113] Transfection is carried out according to the supplier's standard protocol, using Lipofectamine® 3000 (ThermoFisher).

[0114] The cells are then either lysed for detection by the conventional Western blot method using an anti-GFP antibody (Roche #11814460001), or visualized directly on a slide under a fluorescence microscope, according to conventional protocols.

[0115] Visualization by western blot and fluorescence microscopy is carried out after 48 h of transfection, according to conventional operating protocols.

[0116] B / Experiment 1

[0117] This experiment is carried out with the Mmil protein produced in E. coli.

[0118] The gene of sequence SEQ ID No: 2 is cloned into the plasmid, to allow the expression by the cells of the Mmil protein of S. pombe, of amino acid sequence SEQ ID No: 1 (complete Mmil protein).

[0119] Schematically, an extract obtained by cell lysis of cells expressing the Mmil protein is placed in the presence of one or other of the 5' biotinylated RNA molecules "WT RNA" (in accordance with the invention) and "mutated RNA" (not in accordance with the invention, negative control), for a period of 15 min, then the RNA molecules (and the proteins fixed on these molecules) are isolated from the medium by means of Dynabead® magnetic beads on which streptavidin is grafted, using the strong capacity of the latter to bind to biotin. The proteins fixed on the beads are eluted, and analyzed by western blot, after separation of the proteins by electrophoresis on SDS-PAGE gel, using the anti-Mmil antibody.

[0120] The results obtained are shown in [Fig.l] for a dilution of 1 / 100th of the cell lysis extract, for the RNA in accordance with the invention "WT RNA". The presence of the Mmil protein is clearly observed on the western blot membrane, and no background noise, demonstrating the specificity of binding of the UNAAAC RNA motif with the Mmil protein.

[0121] The results obtained are shown in [Fig. 2] for a 1 / 10 dilution of the cell lysis extract, for the RNA in accordance with the invention “WT RNA” and for the RNA not in accordance with the invention “mutated RNA”. Revelation by the anti-Mmil antibody reveals a high intensity band corresponding to the molecular weight of the Mmil protein (54 kDa) when the RNA has a UNAAAC motif in accordance with the invention (“WT RNA”). No band at the molecular weight of the Mmil protein is observed when the “mutated RNA” RNA molecule was used, which demonstrates the binding specificity of the Mmil protein for the UNAAAC motif.

[0122] This experiment clearly demonstrates that the Mmil protein can be purified with a high degree of purity from a complex cellular medium by interaction with an RNA molecule comprising a UNAAAC motif grafted onto a solid support.

[0123] B / Experiment 2 - comparison with immunoprecipitation by antibodies

[0124] This experiment is carried out with the endogenous Mmil protein of S. pombe, amino acid sequence SEQ ID No: 1 (full-length Mmil protein).

[0125] Schematically, an extract obtained by cell lysis of cells expressing the Mmil protein is brought into contact for 1 h with an anti-Mmil antibody.

[0126] After separation of the medium using Sepharose® beads onto which protein A is grafted, the proteins fixed on the beads are eluted and analyzed by western blot, after separation of the proteins by electrophoresis on SDS-PAGE gel, using the anti-Mmil antibody.

[0127] The results obtained are shown in [Fig.3]. We clearly observe, on the track associated with the proteins captured by immunoprecipitation with the anti-Mmil antibody (track "anti-Mmil"), a band corresponding to the molecular weight of the Mmil protein, which is not found on the other tracks. However, we also observe a very high intensity band corresponding to contamination by IgG present in the initial cell extract (band indicated by an arrow on the right in the figure).

[0128] In comparison with this purification by the anti-Mmil antibody, the method according to the invention as implemented in experiment 1, the result of which is illustrated in [Fig.l], allows, four times more quickly (15 min versus 1 h), to purify the Mmil protein with a much higher degree of purity.

[0129] C / Experiment 3 - comparison with TAP immunoprecipitation

[0130] This experiment is performed with the endogenous Mmil protein of S. pombe fused to TAP.

[0131] Schematically, an extract obtained by lysis of cells expressing the Mmil protein in fusion with protein A is placed in contact for 1 h with Sepharose® beads onto which IgG are grafted, in order to isolate the Mmil protein from the medium by taking advantage of the high affinity of IgG for protein A (Kd > 109 M).

[0132] After separation of the beads from the medium, the proteins fixed on the beads are eluted, and analyzed by western blot, after separation of the proteins by electrophoresis on SDS-PAGE gel, using the anti-Mmil antibody.

[0133] The results obtained are shown in [Fig.4]. We clearly observe, on the track associated with the proteins captured by immunoprecipitation with IgG (track "Mmil-TAP"), a band corresponding to the molecular weight of the Protein A - Mmil fusion protein.

[0134] In comparison with this purification by the “Protein A - IgG” system, the method according to the invention as implemented in experiment 1, the result of which is illustrated in [Fig.l], makes it possible, four times more quickly (15 min versus 1 h), to purify the Mmil protein with an equivalent degree of purity, and even slightly higher, in particular because it avoids any contamination with IgG.

[0135] D / Experiment 4 - fusion with the GFP protein

[0136] This experiment is carried out with the Mmil protein produced in HEK293 cells, in fusion with the GFP protein.

[0137] For this purpose, the gene of sequence SEQ ID No: 2 is cloned into the plasmid pEGFP-C3 or pEGFP-N3, to allow the expression by HEK293 cells of the Mmil protein (complete) in fusion, at the C-terminal or at the N-terminal, with the GFP protein. A GFP control alone is also produced.

[0138] The cells obtained are analyzed by western blot after cell lysis or observed by fluorescence microscopy to verify the localization of GFP.

[0139] The results obtained are shown in [Fig.5] for the western blot analysis after cell lysis and in [Fig.6] for the fluorescence analysis of the unlysed cells.

[0140] These results demonstrate that the fusion protein of GFP with Mmil does not present no toxicity for human cells, that it is well expressed and localized in the cells. The results of the Western blot analysis confirm that the sizes of the proteins detected correspond well to those expected (30 KDa for GFP alone and 84 KDa for the Mmil-GFP fusion protein), demonstrating the feasibility of the method according to the invention in mammalian cells.

Claims

Claims

1. A method for purifying a protein of interest, characterized in that it comprises, successively: - the preparation of a fusion protein comprising said protein of interest fused to a protein tag, said protein tag comprising at least: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of said Mmil protein comprising at least the 173 C-terminal amino acids, or a protein with an amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif of nucleotide sequence UNAAAC, - bringing said fusion protein into contact with a ribonucleic acid molecule containing at least one motif of nucleotide sequence UNAAAC, so as to allow the affinity binding of said protein tag with said ribonucleic acid molecule,said ribonucleic acid molecule being grafted onto a solid support or coupled to a capture ligand, - where appropriate, bringing said ribonucleic acid molecule coupled to a capture ligand into contact with an affinity partner of said capture ligand grafted onto a solid support, - and separating said protein of interest from said solid support.,

2. A purification method according to claim 1, wherein an enzymatic cleavage site is inserted between said protein of interest and said protein tag.

3. Purification method according to claim 2, according to which the separation of said protein of interest and said solid support is carried out by cleavage at said enzymatic cleavage site.

4. A purification method according to claim 1 or 2, wherein a cleavage site is inserted between said ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif and said solid support or said ligand of interest, and the separation of said protein of interest and said solid support is carried out by cleavage at said cleavage site.

5. Purification method according to any one of claims 1 to 4, wherein said solid support is a chromatography support, in particular a crosslinked polymer based on polysaccharide or poly- acrylamide.

6. A purification method according to any one of claims 1 to 5, wherein said ribonucleic acid molecule contains one or more repeats of said UNAAAC sequence motif.

7. A purification method according to any one of claims 1 to 6, wherein said ribonucleic acid molecule comprises at least one chemically modified nucleotide and / or at least one blocked nucleic acid and / or at least one phosphorothiate internucleotide linkage.

8. A purification method according to any one of claims 1 to 7, wherein said Mmil protein is derived from a species selected from Schizosaccharomyces pombe, Schizosaccharomyces japonicus, Schizosaccharomyces octosporus and Schizosaccharomyces cryophilus.

9. A method according to any one of claims 1 to 8, wherein said fragment of said Mmil protein comprising at least the C-terminal 173 amino acids has an amino acid sequence comprising, or consisting of, the amino acid sequence SEQ ID No: 9, SEQ ID No: 10, SEQ ID No: 11 or SEQ ID No:

12.

10. A method according to any one of claims 1 to 9, wherein said protein tag contains at least one domain selected from the amino acid sequence domains: - RSVWXaa1Xaa2Xaa3Xaa4Xaa5Xaa6P (SEQ ID No: 13), where Xaaj represents a threonine, serine or alanine residue, Xaa2 represents a threonine, arginine, lysine or serine residue, Xaa3 represents a histidine or arginine residue, Xaa4 represents a threonine or proline residue, Xaa5 represents a glycine, alanine or arginine residue and Xaa6 represents a glutamic acid or aspartic acid residue, - FXaa7SPLKRXaa8APXaa9SXaaioXaaiiXaai2Xaai3Xaai4Xaai5R (SEQ ID No: 14), where Xaa7 represents a serine or threonine residue, Xaa8 represents a proline or glycine residue, Xaa9 represents a glutamic acid or aspartic acid residue, Xaai0 represents a histidine, arginine or lysine residue, Xaan represents an aspartic acid or glutamic acid residue, Xaa[2 represents an alanine or tyrosine residue,Xaan is zero or represents a proline residue, XaaM represents an isoleucine or methionine residue and or tyrosine, Xaa[7 represents a glutamic acid or aspartic acid residue, Xaa[8 represents a leucine or valine residue, Xaa[9 is zero or,

11.

12. represents a serine residue, Xaa20 represents a leucine or methionine residue and Xaa2i represents an arginine, leucine or methionine residue, - QPPXaa22KRRTLXaa23Xaa24P (SEQ ID No: 16), where Xaa22 represents a proline, serine or leucine residue, Xaa23 represents a serine or leucine residue and Xaa24 represents a proline or serine residue, - Xaa25AXaa26Xaa27SPXaa28Xaa2QXaa30Xaa3iPXaa32Xaa33H (SEQ ID No: 17), where Xaa25 represents an arginine or aspartic acid residue, Xaa26 represents a serine or glycine residue, Xaa27 represents a histidine or aspartic acid residue, Xaa28 represents a serine, glycine or leucine residue, Xaa29 represents a leucine or phenylalanine residue, Xaa30 represents a leucine, isoleucine or serine residue, Xaa3i represents a glutamic acid or aspartic acid residue, Xaa32 represents a tyrosine or threonine residue and Xaa33 represents an alanine or threonine residue, - RXaa34EKPKXaa35RAXaa36TPPP (SEQ ID No: 18), where Xaa34 represents a lysine or arginine residue, Xaa35 represents an alanine, proline or threonine residue and Xaa36 represents a serine or proline residue.Solid support for implementing a method for purifying a protein of interest according to any one of claims 1 to 10, characterized in that a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif is grafted onto said solid support. Kit for implementing a method for purifying a protein of interest according to any one of claims 1 to 10, characterized in that it contains a ribonucleic acid molecule containing at least one UNAAAC nucleotide sequence motif grafted onto a solid support or coupled to a capture ligand and at least one of the following constituents: - an expression vector comprising, under the control of a promoter, a nucleic acid molecule coding for a protein tag comprising at least: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of said Mmil protein comprising at least the 173 C-terminal amino acids, or a protein with an amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif of nucleotide sequence UNAAAC; and a site allowing the insertion, 5' or 3' relative to said nucleic acid molecule coding for said protein tag, of a nucleic acid molecule coding for said protein of interest so as to allow the expression of a fusion protein containing said protein of interest and said protein tag; - instructions for implementing the steps of a method according to any one of claims 1 to 10.

13. Kit according to claim 12, further containing a host cell capable of being transformed by an expression vector comprising, under the control of a promoter, a nucleic acid molecule encoding a protein tag comprising at least: the Mmil protein of a microorganism of the genus Schizosaccharomyces, a fragment of said Mmil protein comprising at least the C-terminal 173 amino acids, or a protein of amino acid sequence having at least 90% identity with the amino acid sequence of said Mmil protein or said fragment and capable of binding to a ribonucleic acid motif of nucleotide sequence UNAAAC;and a site allowing the insertion, 5' or 3' relative to said nucleic acid molecule coding for said protein tag, of a nucleic acid molecule coding for said protein of interest so as to allow the expression of a fusion protein containing said protein of interest and said protein tag.;

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