ES2800925T3 - Diagnostic use of a fusion polypeptide comprising a viral protein and an MGMT enzyme - Google Patents

Abstract

In vitro use of a fusion polypeptide comprising a viral protein and i) the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63) or a homologue thereof, whose sequence has an identity of at least 70% with SEQ ID NO: 4, or ii) the SNAP mutant of SEQ ID NO: 2 or a homolog thereof, whose sequence exhibits an identity of at least 80% with SEQ ID NO: 2, as a diagnostic agent for detection of a viral infection in a biological fluid of a patient.

Description

DESCRIPTION

Diagnostic use of a fusion polypeptide comprising a viral protein and an MGMT enzyme

The present invention relates to the field of genetic engineering and molecular biology. In particular, the present invention discloses a new enhancer of protein production in host cells. Furthermore, the present invention discloses vectors that contain the DNA sequence coding for said enhancer protein and also the use of the same for the expression of recombinant proteins, such as industrial enzymes or proteins for pharmaceutical use, including eukaryotic proteins (for example of mammalian, such as human) and viral proteins.

Background of the invention

Protein production systems, in which polypeptides or proteins of interest are produced in recombinant organisms or cells, are the backbone of commercial biotechnology.

The first systems, based on bacterial expression in hosts such as E. coli, have been followed by systems based on eukaryotic hosts, in particular mammalian cells in culture, insect cells both in culture and in the form of whole insects, and mammals. transgenic such as sheep and goats.

Prokaryotic cell culture systems are easy to maintain and inexpensive to operate. However, prokaryotic cells are not capable of post-translationally modifying eukaryotic proteins. Furthermore, many proteins fold incorrectly, requiring specific procedures to fold them again, which adds to the cost of production.

Eukaryotic cell culture systems have been described for various applications. For example, some mammalian cells are capable of post-translational modifications and generally produce proteins that are correctly folded and soluble. Major disadvantages of mammalian cell systems include the need for specialized and expensive culture facilities, the risk of infection, which can lead to loss of the entire culture, and a risk of contamination of the end product with potentially mammalian proteins. dangerous. Insect cells are alternatively used for the expression of polypeptides. The most widespread expression system used in insect cells is based on baculovirus vectors. A baculovirus expression vector is constructed by replacing the baculovirus polyhedrin gene, which encodes a larger baculovirus structural protein, with a heterologous gene, under the control of the strong native polyhedrin promoter. The insect host cells in culture become infected by the recombinant virus and the protein produced in this way can be recovered from the cells themselves or from the culture medium in case suitable secretion signals are used.

However, both systems suffer from problems associated with the reproducibility of the level and quality of expression of the recombinant proteins, the infection of the culture and the fact that they may require specialized culture facilities. Furthermore, the baculovirus stocks, which for the production of certain proteins might require preparation under GMP conditions, are not stable over time in all cases.

Drosophila cells , in particular Drosophila melanogaster S2 cells, for protein expression have been disclosed in US Patent Nos. 5,550,043, 5,681,713 and 5,705,359. In contrast to the prior art baculovirus system, in which the protein of interest is provided only after lysis of infected insect cells, the S2 cell-based method provides a continuous cellular expression system for heterologous proteins and, therefore, it leads to higher expression levels.

Some other means have been proposed to enhance the expression of heterologous proteins in host cells: for example, US Patent No. 5,919,682 describes a method of overproduction of functional nitric acid synthase in a prokaryote using a pCW vector under the control of the promoter tac and protein co-expression with chaperones. In addition, US patent No. 4,758,512 refers to the production of host cells that present specific mutations within their DNA sequences that cause the organism to show a reduced capacity for degradation of foreign products. These mutated host organisms can be used to increase the yields of genetically engineered foreign proteins.

Vertebrate cells, in particular mammalian cells, have also been widely used in the expression of recombinant proteins. The level of protein production during the time of the cells in culture depends on several factors, such as, for example, the cell density, the phase of the cell cycle, the cellular biosynthesis rates of the proteins, the condition of the medium used for cell viability and growth; and longevity of cells in culture (ie, how long it takes before they succumb to programmed cell death, or apoptosis). Various methods have been developed to improve the viability and lifespan of cells in culture, in conjunction with methods of increasing the productivity of a desired protein by, for example, controlling nutrients, cell density, content of oxygen and carbon dioxide, lactate dehydrogenase, pH, osmolarity, catabolites, etc.

Other host cells can be used to produce heterologous recombinant proteins, notably plant cells and yeast cells.

Many pharmaceutical proteins of mammalian origin have been synthesized in plants. These include blood products, such as human serum albumin, for which there is an annual demand in excess of 500 tons, and cytokines and other signaling molecules that are needed in much smaller quantities. Most plant-derived proteins have been produced in transgenic tobacco plants and have been extracted directly from the leaves. In general these proteins are produced at low levels, typically less than 0.1. of the total amount of soluble proteins. This low level of production likely reflects a combination of factors, the most important being poor protein folding and stability. More recently, the tobacco chloroplast system has been used to express human proteins at much higher levels (MA JKC et al., 2004).

Yeast systems have been instrumental in producing large amounts of protein for industrial and biopharmaceutical use for many years. Yeasts can be grown to very high cell mass densities in a well-defined medium. Recombinant proteins in yeast can be overexpressed such that the product is secreted from the cell and is available for recovery in the fermentation solution. Yeast secreted proteins are strongly glycosylated at consensus glycosylation sites. Thus, the expression of recombinant proteins in yeast systems has historically been restricted to proteins in which posttranslational patterns of glycosylation do not affect protein function. Several yeast expression systems are used for the expression of recombinant proteins, including Saccharomyces, Schizosaccharomyces pombe, Pichia pastoris, and Hansanuela polymorpha. Recently a new system has appeared with the ability to produce recombinant glycoproteins in yeast that has glycosylation sequences similar to those of secreted human glycoproteins that are produced in mammalian cells. The glycosylation pathway of Pichia pastoris was modified by eliminating endogenous enzymes, which add chains rich in mannose to N-glycosylation intermediates. Furthermore, at least five active enzymes participating in the synthesis of humanized oligosaccharide chains were specifically transferred into P. pastoris . The ability to produce large amounts of humanized glycoproteins in yeast offers the advantage that glycosylated structures can be very uniform and easy to purify. Furthermore, cross-contamination by mammalian viruses and glycoproteins from other mammalian hosts can be eliminated by using semi-continuous production in yeast, with fermentation times shorter than in mammalian cells.

However, using such systems, heterologous proteins are produced at a rate of approximately 1 to 2 mg / L in the supernatant of cultured cells, which is a fairly low level for industrial production purposes.

Thus, there is an urgent need to provide a system that allows achieving a significantly high level of expression of heterologous proteins.

The present application satisfies this need and provides protein expression methods that achieve a production level up to 100 times higher than existing means of protein production (ie, up to 200 mg / L protein in the supernatant).

The present inventors have indeed demonstrated that the use of a nucleotide vector encoding a protein derived from the human protein 6-methylguanine-DNA methyltransferase (MGMT_h), said derived protein being linked MGMT_h, directly or not, to a protein of interest increases the production of said protein of interest up to a yield of 40 mg / l to 200 mg / l on average.

More specifically, the invention relates to the following points:

1. In vitro use of a fusion polypeptide comprising a viral protein and

- the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63) or a homologue thereof, the sequence of which has an identity of at least 70% with SEQ ID No: 4, or

- the SNAP mutant of SEQ ID No: 2 or a homolog thereof, whose sequence has an identity of at least 80% with SEQ ID No: 2,

as a diagnostic agent for the detection of a viral infection in a biological fluid of a patient.

2. Use according to claim 1, wherein said viral protein is EDIII protein from chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), yellow fever (YF), Usutu ( USU) or West Nile, or nucleoprotein N from Rift Valley or Tuscany fever viruses.

3. Use according to claim 1 or 2, wherein said biological fluid is urine, blood, serum or saliva.

4. A diagnostic immunoassay method using the fusion polypeptide as defined in claims 1 to 2 and a biological fluid from a patient.

5. A diagnostic immunoassay method according to claim 4, wherein said fusion polypeptide is coupled to beads.

6. A diagnostic immunoassay method according to claim 4, wherein said fusion polypeptide is coupled to beads using a substrate of the MGMT protein as a linker.

7. A diagnostic immunoassay method according to any one of claims 4 to 6, for rapidly and simultaneously detecting antibodies in a biological liquid.

8. A diagnostic immunoassay method according to claim 7, wherein said biological fluid is urine, blood, serum or saliva.

9. Diagnostic kit containing the fusion polypeptide as defined in claim 1, wherein said viral protein is a protein from chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE) viruses, Yellow fever (YF), Usutu (USU), West Nile, Rift Valley fever or Tuscany.

10. Use of the diagnostic kit according to claim 9 to identify a viral infection in a biological sample from a patient.

11. Use according to claim 10, wherein said biological sample is urine, blood, serum or saliva.

Legends of the figures

Figure 1 discloses (A) a schematic view of the mRNA encoding a MGMT fusion protein sequence of the application, containing, from 5 'to 3', a signal peptide, the MGMT mRNA sequence, a spacer, a protease cleavage site, a recombinant protein gene (foreign gene), a spacer and a tag (His6), and (B) the DNA and amino acid sequences of the same part of the vector, comprising : i) the insect single-stranded BiP signal peptide (BiPmc) (in italics), ii) the SNAP-encoding enhancer sequence (in gray), iii) a DNA spacer sequence, iv) the DNA-site coding sequence enterokinase (in bold), v) the EcoRV / Xmal cloning sites (underlined) and vi) the DNA encoding the His tag tag (italics and bold) (see also SEQ ID No. 5).

Figure 2 discloses (A) the amino acid sequence of the SNAP fusion protein (in gray) and Rift Valley fever virus nucleoprotein N (FVR.N., in bold) attached to a label of His tag, separating both proteins with a GGGS spacer, (B) immunoblotting assay in cell supernatant of S2 cells transfected with the vector DNA of SEQ ID No. 19 (SNAP-FVR) stimulated or not with cadmium for 10 days, using anti-Hislabel antibodies, and (C) an immunoblot assay carried out with anti-His antibodies, on insoluble (INS) or soluble (SOL) protein fractions of E. coli B21 lysates, said bacteria carrying a plasmid pET302 /FVR.N+proTEV+GST. (D) An immunoblot assay showing the amount of s Na P-FVR.N in successive fraction samples, obtained after a two-step purification of secreted chimeric proteins SNAP-FVR.N from S2 cells stimulated for 10 days, using Talon and Superdex 75 columns.

Figure 3 discloses (A) the DNA and amino acid sequences of the SNAP fusion protein (in italics) and the soluble form of the West Nile virus coat protein E (in gray), attached to a label His tag (in bold), the proteins being separated by a GGGS spacer (SEQ ID No. 20) and (B) immunoblot assay with anti-His tag antibodies, showing the secretion of the soluble form of the His tag protein. E coat of the West Nile virus in the supernatant of S2 cells transfected with the DNA vector of the present application encoding SNAP-WNsE (SEQ ID No. 20) and stimulated or not with cadmium for 10 days.

Figure 4 discloses (A) a schematic of the DNA cassette containing a BiP signal peptide, a coding sequence of the MGMT type (SNAP type), two pro-TEV (tobacco etch virus protease) cleavage sites ) on each side of the IFNa sequence (IFNAI_hu) and a His tag mark, (B) the DNA and amino acid sequences of the SNAP fusion protein (in gray, preceded by an insect peptide signal, in italics ) and IFNa (in bold), followed by His tag marking (in bold and italics), the SNAP and IFNa proteins being separated by the enterokinase cleavage site (underlined) and a Gg S spacer sequence (C ) Immunoblotting assay with anti-His tag antibodies, to detect the expression of IFNa in the supernatant of S2 cells transfected with the vector of the present application encoding IFNa (S2 / SNAP-IFN) or a control vector, stimulated or not with Cd2 +. (D) Immunoblotting assay with anti-SNAP antibodies in 10 µl supernatant of S2 / DeSNAPuniv-IFNa cells induced or not induced for 10 days with cadmium, (E) luciferase activity in HeLa cells infected by Chikungunya virus expressing a Renilla luciferase , where said cells are treated with different doses of iFNa, from a commercial supplier (Intergen), or with the IFNa produced by the method of the present application. (F) Luciferase activity in HeLa cells infected by Chikungunya virus expressing a Renilla luciferase , wherein said cells are treated with different doses of the SNAP-IFNa protein obtained by the production method described in the present application.

Figure 5 represents the different stages of the recombinant protein production process described in the present application.

Figure 6 discloses (A) the DNA and amino acid sequences of the SNAP fusion protein (in gray, preceded by an insect peptide signal) and granzyme M, followed by a His-tag marking, the SNAP and granzyme M proteins by the enterokinase cleavage site and a GGGS spacer sequence. (B) Schematic view of the SNAP-GrM chimeric fusion protein, underlining the three potential GrM protease cleavage sites in SNAP. (C) Immunoblot assay with anti-SNAP and anti-His tag antibodies for the detection of SNAP-GrM expression in the supernatant of S2 cells transfected with the vector of the present application encoding GrM (S2 / SNAP-GrM, SEQ ID No. 55).

Figure 7 discloses (A) a scheme of the universal DNA cassette containing a BiP-like signal peptide, an MGMT coding sequence, two pro-TEV cleavage sites on each side of the IFNa sequence (IFNAI_hu) and a His-tag tag, (B) the DNA and amino acid sequences of the SNAP fusion protein (in gray, preceded by an insect BiP-like peptide signal) and human IFNal (amino acids in bold), followed by a tag His tagging, the SNAP and IFNa proteins being separated by the pro-TEV cleavage site and a GGGS spacer sequence. (C) Immunoblot assay with anti-SNAP antibodies for detection of SNAP-IFNa expression in the supernatant of HeLa cells transfected with a vector encoding SNAP alone without signal peptide (vector pSNAPf) or a vector encoding SNAP alone, preceded by the Dengue virus peptide signal (pDV1ssprM-SNAP) or the vector of the present application encoding IFNa, which comprises the DNA sequence as defined in (A) (pDeSNAP-4 / SNAP-IFNA1, SEQ ID n 57).

Figure 8A discloses a universal DNA cassette containing a BiP-like signal peptide, a SNAP coding sequence, two pro-TEV cleavage sites, a His tag tag, four unique cloning sites, BamHI, EcoRV, XmaI and ApaI for the cloning of a gene of interest and a GGGS spacer sequence (DeSNAP Univ, SEQ ID No. 59 and No. 60). The unique Nh3I sites at the 5 'end and Notl / HindIII at the 3' end are necessary for the subcloning step in mammalian expression vectors (for example the plasmid pcDNA3 or pCI-neo) and the unique Bgl II sites in the 5 'end and Age I at the 3' end are required for the subcloning step in the non-vertebrate DES system. The scheme in (B) discloses the universal DNA cassette containing a BiP-like signal peptide, an MGMT coding sequence, two pro-TEV cleavage sites, a His tag tag, four unique cloning sites : BamHI, EcoRV, Smal and ApaI for the cloning of a gene of interest and a GGGS spacer sequence (DeMGMT univ, SEQ ID No. 69 and No. 70).

Figure 9 discloses a means of inserting a foreign gene of interest into DeMGMT Univ.

Figure 10 shows the thermal stability of SNAP fusion proteins called CHIK.sE2-SNAP, SNAP-WN.EDIII and SNAP-IFNaI incubated for 4 days at -80 ° C, at 4 ° C, at 25 ° C or 37 ° C (A) or for two months at -80 ° C, 4 ° C, 25 ° C or 37 ° C (B).

Figure 11 shows the production of the fusion proteins SNAP-SSX2 and SNAP-sFasL by the vectors of the present application introduced into S2 cells, after 10 days of induction with cadmium (+) or without cadmium (-) in supernatant complete (A) or in different fractions (B).

Figure 12 discloses (A) a scheme of the universal DNA cassette containing a BiP-like signal peptide, a coding sequence for MGMT (SNAP-like), two pro-TEV cleavage sites, on each side of the antigen SSX2 cancer cell and a His tag tag, (B) a universal DNA cassette scheme containing a BiP-like signal peptide, an MGMT coding sequence, two pro-TEV cleavage sites on each side of the NERMCSL protein and a His tag label, (C) an immunoblot assay in HeLa cells transiently transfected for two days using anti-SNAP mouse antibodies, showing extracellular or intracellular production of IFNa, SSX2 and NERMCSL.

Figure 13 discloses (A) a scheme of the universal DNA cassette containing a BiP-like signal peptide, an MGMT (SNAP-like) coding sequence, two pro-TEV cleavage sites on each side of the SULF_h polypeptide. -2ñTMD, and a His tag mark, (B) the DNA and amino acid sequences of the SNAP fusion protein (in dark gray, preceded by an insect BiP-like peptide signal) and hSULF2 ñTMD, followed by a Histag label, the SNAP and SULF_h-2ñTMD proteins being separated by the pro-TEV cleavage site and a GGGS spacer sequence and (C) the enzymatic activity of chimeric DeSNAP-SULF_h-2ñTMD secreted by the cells HEK 293 transiently transfected for two days with pcDNA3 / DeSNAPuniv-SULF_h-2ñTMD

Figure 14 discloses (A) a schematic of the DNA cassette containing a BiP signal peptide, an MGMT (SNAP-like) coding sequence, two pro-TEV cleavage sites on each side of the NERMCSL protein, and a His tagging, and (B) an immunoblot assay using anti-SNAP antibodies, for the detection of the expression of the NERMCSL protein in the supernatant of S2 cells transfected with the vector of the present application encoding the NERMCSL protein ( S2 / SNAP-NERMCSL) or with a vector encoding the soluble protein E2 of the Chikungunya virus (CHIK.sE2-SNAP) stimulated or not with Cd2 +.

Detailed description of the invention

In the context of the present invention it is appreciated that the co-expression of the enzyme 6-methylguanine-DNA-methyltransferase (MGMT) together with a recombinant protein of interest greatly improved the production of said recombinant protein in insect cells, such as S2 cells, as well as in mammalian cells, such as in HeLa cells.

The enzyme 6-methylguanine-DNA-methyltransferase (MGMT, also known as ATase or AGT, and hereinafter referred to as "MGMT") has the reference EC 2.1.1.63 in the IUBMB enzyme nomenclature. It is a DNA repair enzyme 6-alkylguanine-DNA-alkyltransferase of 207 amino acid residues, the function of which in cells is to repair alkylated DNA. More precisely, MGMT acts on the O6-methylated guanine in DNA by transferring the methyl group in a Sn2 reaction to a reactive cysteine residue (Cys 145). The repair mechanism is unusual in that the protein is irreversibly inactivated (Pegg AE et al., Mutat. Res. 462: 82-100, 2000). This enzyme is currently used in molecular biology for the labeling of proteins in vivo with reporter molecules, through an irreversible labeling reaction with O6-benzylguanine derivatives (Juillerat A. et al., Chemistry & Biology 10: 313-317, 2003 and document No. WO 2005/085470).

So far, different enzymes derived from MGMT have been described (Lim A. et al., EMBO J. 15: 4050-4060, 1996; Daniels DS et al., EMBO J. 19: 1719-1730, 2000, Juillerat A. et al., Chemistry & Biology 10: 313-317, 2003, documents No. WO 2005/085470, No. WO 2004/031405). In particular, a 20 kDa mutant protein has been obtained that contains the mutations Cys62Ala, Lys125Ala, Ala127Thr, Arg128Ala, Gly131Lys, Gly132Thr, Met134Leu, Arg135Ser, Cys150Ser, Asn157Gly, Ser159Glu "182 truncated in the amino acid" AGT26 document No. WO 2005/085470, also referred to as "SNAP 26" in document No. WO 2006/114409). The particular mutant "SNAP26" has been shown to exhibit increased labeling activity. However, it has never been shown or suggested that it can increase the expression of the recombinant proteins to which it is coupled.

Herein the present inventors propose for the first time the use of the enzyme MGMT (EC 2.1.1.63), a mutant, a catalytic domain thereof or sub-fragments thereof, to increase protein production in host cells, in particular in non-vertebrate and vertebrate host cells. The potentiating effect is observed when the host cells express a fusion polypeptide comprising at least: i) a secretion signal peptide that is functional in said host cells, ii) the MGMT enzyme, mutant, catalytic domain or sub-fragments thereof , and iii) the protein of interest. For the potentiating effect to occur, the MGMT enzyme must physically bind, directly or indirectly (spacers and other amino acids can be introduced), to the protein of interest. Without being bound by any theory, it is contemplated that the MGMT enzyme may serve as a chaperone protein, for example by promoting secretion from the host cell and stabilizing the synthesized fusion polypeptide in the host cell supernatant, or preventing it from being metabolized. during and after its synthesis and secretion from host cells.

Furthermore, MGMT has been found to exhibit a 3D globular structure comprising an a-helix (Wibley JEA et al., 2000) that is compatible with a scaffold function of MGMT.

In the context of the present application, "host" cells are any cells that can be used to produce recombinant proteins, such as "non-vertebrate" (or invertebrate) cells, vertebrate cells, plant cells, yeast cells or cells. prokaryotic. They are preferably non-vertebrate and vertebrate cells.

Non-vertebrates (also known as invertebrates) comprise different divisions, the best known of which are: Insects, Arachnids, Crustaceans, Mollusks, Annelids, Cirripeds, Radiated, Coelenterates and Infusorians. They are currently classified into more than 30 divisions, from simple organisms such as sea sponges and flatworms, to complex animals such as arthropods and mollusks. In the context of the present application, the non-vertebrate cells are preferably insect cells, such as Drosophila or Mosquito, more preferably Drosophila S2 cells.

Examples of cells derived from vertebrate organisms that are useful as host cell lines include non-human embryonic stem cells or derivatives thereof, for example avian EBX cells, the monkey kidney CVI line transformed by SV40 sequences (COS -7, ATCC # CRL 1651), human embryonic kidney line (293), neonate hamster kidney cells (BHK, ATCC # CCL 10), Chinese hamster ovary (CHO) cells, mouse Sertoli cells [TM4], monkey kidney cells (CVI, ATCC # CCL 70), African green monkey kidney cells (VERO-76, ATCC # CRL-1587), human cervical carcinoma cells (HeLa, ATCC # CCL 2 ), canine kidney cells (MDCK, ATCC n ° c Cl 34), buffalo rat liver cells (BRL 3A, ATCC n ° CRL l442), human lung cells (W138, ATCC n ° CCL 75), human liver cells (Hep G2, HB 8065), mouse mammary tumor cells (MMT 060562, ATCC # CCL51), rat hepatoma cells [HTC, Ml.5], and YB2 / O cells (ATCC # CRL1662), NIH3T3, HEK and TRI. In the context of the present application, the vertebrate cells are preferably EBX, CHO, YB2 / O, COS, HEK, NIH3T3 cells or derivatives thereof.

Plant cells that can be used in the context of the present application are Bright Yellow 2 (BY2) and Nicotiana tabaccum 1 (NT-1) tobacco cultivars.

Yeast cells that can be used in the context of the present application are: Saccharomyces cerevisiae, Schizosaccharomyces pombe and Hansenula polymorpha, as well as methylotrophic yeasts such as Pichia pastoris and Pichia methanolica.

Prokaryotic cells that can be used in the context of the present application are typically E. coli bacteria or Bacillus subtilis bacteria .

The present application thus discloses a vector for the expression of nucleotides encoding at least a) a peptide secretion signal, which is preferably functional in non-vertebrate cells or in vertebrate cells, and b) a 6-methylguanine-DNA-methyltransferase enzyme , a mutant, a sub-fragment or a catalytic domain thereof.

The term "vector" herein refers to the vehicle into which a DNA or RNA sequence of a foreign gene can be introduced into a host cell in a manner that is transformed and the expression of the introduced sequence is promoted. Vectors can include, for example, plasmids, phages, and viruses and are discussed in more detail below. Indeed, any type of plasmid, cosmid, YAC or viral vector can be used to prepare a recombinant nucleic acid construct that can be introduced into a host cell in which expression of the protein of interest is desired. Alternatively, in the event that expression of the protein of interest is desired in a particular type of host cell, viral vectors can be used that selectively infect the desired cell type or tissue type. Also important in the context of the present application are vectors for use in gene therapy (that is, those capable of transporting the nucleic acid molecule to a host organism).

For example, viral vectors, such as lentiviruses, retroviruses, herpes viruses, adenoviruses, adeno-associated viruses, vaccinia viruses, baculoviruses, and other recombinant viruses with desirable cell tropism. Methods for the construction and use of viral vectors are known in the art (see Miller and Rosman, BioTechniques 7: 980-990, 1992).

Viral vectors that are currently preferred in the present application are those that are suitable for use in vertebrate and non-vertebrate cells.

For non-vertebrate cells, the preferred vectors are arboviruses, with West Nile virus being particularly preferred, which are arthropod vectors. Other vectors that are known to be efficiently expressed in non-vertebrate cells are baculoviruses.

For vertebrate cells, lentiviral, AAV, baculoviral and adenoviral vectors are preferred. Vectors suitable for expression in mammalian host cells can also be of non-viral origin (eg plasmid DNA). Suitable plasmid vectors include, but are not limited to, pREP4, pcEP4 (Invitrogene), pCI (Promega), pCDM8, and pMT2PC, pVAX, and pgWiz.

For prokaryotic cells, plasmid, bacteriophage, and cosmid vectors are preferred. Vectors suitable for use in prokaryotic systems include, but are not limited to, the vectors pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pPoly, pTrc, pET 11d, pIN, and pGEX.

For plant cells, plasmid expression vectors, such as Ti plasmids, and viral expression vectors, such as cauliflower mosaic virus (CaMV) and tobacco mosaic virus, are preferred. TMV.

The expression of recombinant proteins in yeast cells can be carried out using three types of vector: integration vectors (YIp), episomal plasmids (YEp) and centromeric plasmids (YCp). Suitable vectors for expression in yeast (e.g. S. cerevisiae) include, but are not limited to, pYepSec1, pMFa, pJRY88, pYES2 (Invitrogen Corporation, San Diego, Calif.), And pTEF-MF (Dualsystems Biotech product code P03303).

Vectors that can be used for gene therapy are well known in the art. They are, for example, lentivirus, retrovirus, adenovirus, poxvirus, herpes virus, measles virus, foamy virus or adeno-associated virus (AAV). Viral vectors can be replication competent or they can be genetically disabled such that they are replication defective or replication impaired. Preferred gene therapy vectors are DNA overlapping vectors ("DNA flap"), as indicated in document No. WO 1999/055892, US patent No. 6,682,507 and document No. WO 2001/27300 .

A "coding" sequence for an expression product, such as an RNA, polypeptide, protein, or enzyme, is a nucleotide sequence that, when expressed, results in the production of said RNA, polypeptide, protein, or enzyme; that is, the nucleotide sequence "encodes" said RNA or encodes the amino acid sequence for said polypeptide, protein, or enzyme.

In the context of the present application, the "catalytic domain" of an enzyme refers to the active site of the enzyme or, in other words, the part of an enzyme molecule in which catalysis of the substrate occurs (herein , the transfer of the methyl group in a Sn2 reaction to a reactive cysteine residue). Therefore, the term "a catalytic domain thereof" refers to any homologous fragment or sequence of the MGMT polypeptide, preferably exhibiting a catalytic activity of at least 80% that of the native MGMT enzyme. These fragments (also called "sub-fragments") can comprise between 20 and 180, preferably between 30 and 100 amino acids. The homologous sequence of said catalytic domain can present one or more mutations, resulting in the partial or total loss of said catalytic activity.

In the context of the invention, the MGMT enzyme can be the human MGMT (with reference NP_002403.2) of sequence SEQ ID No. 4, the mouse MGMT identified as NP_032624.1 (SEQ ID No. 45), the MGMT of rat identified as NP_036993.1 (SEQ ID No. 46) or homologous sequence thereof.

The term "homologous" refers to sequences that exhibit sequence similarity. The term "sequence similarity", in all grammatical forms thereof, refers to the degree of identity or correspondence between nucleic acid or amino acid sequences. In the context of the invention, two amino acid sequences are "homologous" in the event that at least about 80%, alternatively at least about 81%, alternatively at least about 82%, alternatively at least about 83% , alternatively at least about 84%, alternatively at least about 85%, alternatively at least about 86%, alternatively at least about 87%, alternatively at least about 88%, alternatively at least about 89%, alternatively at least about 90%, alternatively at least about 91%, alternatively at least about 92%, alternatively at least about 93%, alternatively at least about 94%, alternatively at least about 95%, alternatively at least about minus about 96%, alternatively at least about 97%, alt Alternatively at least about 98%, alternatively at least about 99% of the amino acids are similar. Preferably, similar or homologous polypeptide sequences are identified using the Needleman and Wunsch algorithm.

Preferably, the sequence homologous to the enzyme 6-methylguanine-DNA-methyltransferase shares an amino acid sequence identity of at least 64%, preferably an amino acid sequence identity of at least 65%, alternatively an amino acid sequence identity of at least about 66%, alternatively an amino acid sequence identity of at least about 67%, alternatively an amino acid sequence identity of at least about 68%, alternatively an amino acid sequence identity of at least about 69%, alternatively an amino acid sequence identity of at least about 70%, alternatively an amino acid sequence identity of at least about 71%, alternatively an amino acid sequence identity of at least about 72%, alternatively a amino acid sequence identity of at least approximate alternatively 73%, alternatively an amino acid sequence identity of at least about 74%, alternatively an amino acid sequence identity of at least about 75%, alternatively an amino acid sequence identity of at least about 76%, alternatively an amino acid sequence identity of at least about 77%, alternatively an amino acid sequence identity of at least about 78%, alternatively an amino acid sequence identity of at least about 79%, alternatively a sequence identity of amino acids of at least about 80%, alternatively an amino acid sequence identity of at least about 81%, alternatively an amino acid sequence identity of at least about 82%, alternatively an amino acid sequence identity of at least about 83%, alternatively an amino acid sequence identity of at least about 84%, alternatively an amino acid sequence identity of at least about 85%, alternatively an amino acid sequence identity of at least about 86%, alternatively an amino acid sequence identity of at least about 87%, alternatively an amino acid sequence identity of at least about 88%, alternatively a sequence identity of amino acids of at least about 89%, alternatively an amino acid sequence identity of at least about 90%, alternatively an amino acid sequence identity of at least about 91%, alternatively an amino acid sequence identity of at least about 92%, alternatively a an amino acid sequence identity of at least about 93%, alternatively an amino acid sequence identity of at least about 94%, alternatively an amino acid sequence identity of at least about 95%, alternatively a sequence identity of amino acids of at least about 96%, alternatively an amino acid sequence identity of at least about 97%, alternatively an amino acid sequence identity of at least about 98%, alternatively an amino acid sequence identity of at least about 99% relative to SEQ ID No. 4. In a preferred embodiment, the homologous sequence of SEQ ID No. 4 is at least 64%, preferably 70%, and more preferably 80% identical to SEQ ID No. Four.

A more preferred homologous MGMT sequence contains the mutations indicated in WO 2005/085470, the positions of which can be easily rearranged in view of SEQ ID No. 4, the starting methionine residue of SNAP26 corresponding to residue of methionine at position 32 of SEQ ID No. 4 (therefore, 31 amino acids should be added to the positions disclosed in document No. WO 2005/085470 in order to obtain the corresponding ones in SEQ ID No. 4).

Preferably, the homologous sequence of MGMT useful in the invention corresponds to the sequence of wild-type MGMT of SEQ ID No. 4, in which between 1 and 30 have been deleted, preferably between 6 and 25, and in particular 14, 15 , 16, 17, 18, 19, 20, 21, 22 or 23 amino acids are substituted by other amino acids, and / or 1 to 40, preferably 1 to 20, in particular 10 to 20 amino acids, more preferably 15 amino acids at the C terminal -terminal.

In a preferred embodiment, the homologous sequence of MGMT contains the following mutations with respect to SEQ ID No. 4:

(A) Lys31 substituted by Arg, or Met32 substituted by Ser, or Cys93 substituted by Ala, or Lys156 substituted by Ala, or Ala158 substituted by Thr, or Arg159 substituted by Ala, or Gly162 substituted by Lys, or Gly163 substituted by Thr, or Met165 substituted by Leu, or Arg166 substituted by Ser, or Cys181 substituted by Ser, or Asn188 substituted by Gly, or Ser190 substituted by Glu, or Gly214 substituted by Pro, or Ser215 substituted by Ala, or Ser216 substituted by Gly, or Gly217 substituted by Ile, or Leu218 substituted by Gly, or Gly220 substituted by Pro, or Ala221 substituted by Gly, or Trp222 substituted by Ser, or

(B) Lys31-Met32 substituted by Arg-Ser, or Ala158-Arg159 substituted by Thr-Ala, r Gly162-Gly163 substituted by Lys-Thr, or Met165-Arg166 substituted by Leu-Ser, or Gly162-Gly163 / Met165-Arg166 replaced by Lys-Thr / Leu-Ser, or Asn188 / Ser190 replaced by Gly / Glu, or Gly214-Ser215-Ser216-Gly217-Leu218 replaced by Pro-Ala-Gly-Ile-Gly, or Gly220-Ala221-Trp222 replaced by Pro-Gly-Ser, preferably in combination with any other amino acid substitutions mentioned in (A), or

(C) Truncated after Leu223 (amino acids 224 to 238 have been deleted), preferably in combination with any other amino acid substitution cited in (A) or (B).

Preferred MGMT homologous sequences are those truncated after Leu223.

Preferred MGMT homologous sequences are those with two of the modifications of (B) and optionally truncated after Leu223.

Preferred MGMT homologous sequences are those with three of the modifications of (B) and optionally truncated after Leu223.

Preferred MGMT homologous sequences are those with four of the modifications of (B) and optionally truncated after Leu223.

Preferred MGMT homologous sequences are those with five of the modifications of (B) and optionally truncated after Leu223.

Preferred MGMT homologous sequences are those with six of the modifications of (B) and optionally with truncate after Leu223.

Other preferred MGMT homologous sequences are those that contain a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mutations selected from the modifications disclosed in (A), and optionally truncated after Leu223.

Particularly preferred are homologous sequences that contain the mutations Lys31Arg, Met32Ser, Cys93Ala, Lys156Ala, Ala158Thr, Arg159Ala, Gly162Lys, Gly163Thr, Met165Leu, Arg166Ser, Cys181Ser, As16148Gly, Serly1Glu2Gly, Gly19u202, Gly19u2Gly2, Gly19u2Pro Trp222Ser and truncated after Leu223 (ie, the SNAP sequence of SEQ ID No. 2).

In an even more preferred embodiment, the MGMT enzyme is the SNAP mutant protein of SEQ ID NO: 2 or a homologue thereof. The SNAP mutant of SEQ ID No. 2 shares a 77% homology with the amino acid sequence of human 6-methylguanine-DNA-methyltransferase (NP_002403.2, SEQ ID No. 4) and a homology of 70% with respect to the amino acid sequence of mouse 6-methylguanine-DNA-methyltransferase (NP_032624.1, SEQ ID NO: 45).

Preferably, said sequence homologous to the SNAP protein is at least identical to more than 80%, preferably to 81%, more preferably to 82%, more preferably to 83%, more preferably to 84%, more preferably to 85%, more preferably 86%, more preferably 87%, more preferably 88%, more preferably 89%, more preferably 90%, more preferably 91%, more preferably 92%, more preferably 93%, more preferably 94%, more preferably 95%, more preferably 96% and still more preferably 97% with respect to the SNAP protein of sequence SEQ ID No. 2.

Preferably, the disclosed nucleotide expression vector further comprises cloning sites that allow insertion into frame of a heterologous DNA sequence encoding a protein of interest.

The term "peptide secretion signal" as used in the present application refers to a short peptide chain (3 to 60 amino acids in length) that directs the transport of a protein.

Examples of appropriate secretion signals for the present vectors include, but are not limited to, alpha mating factor (MF) signal peptide sequences (US Patent No. 5,879,926), invertase (document No. WO84 / 01153), PHO5 (DK patent No. 3614/83), YAP3 (document No. WO95 / 02059) and BAR1 (document No. WO87 / 02670).

In the context of the present application, this peptide secretion signal is preferably functional in non-vertebrate cells or in vertebrate cells, or both.

Examples of peptide secretion signals that are functional in insect cells are: insect BiPmc (SEQ ID No. 48, exhibiting for example the DNA sequence SEQ ID No. 11), the BiP-type peptide signal of SEQ ID No. No. 51 (eg having the DNA sequence SEQ ID No. 50) and any peptide signal present in an arbovirus, for example West Nile virus coat protein E (SEQ ID No. 15).

Interestingly, the above-mentioned BiP-like signal peptide is functional in both non-vertebrate and vertebrate cells. This BiP-like signal corresponds to the BiP peptide signal of SEQ ID No. 48 in which the last glycine amino acid has been replaced by the amino acid sequence Pro Thr Ala Leu Ala (SEQ ID No. 61), which corresponds to the site of Dengue virus E protein cleavage. In accordance with the foregoing, the BiP-like signal is advantageously cut after the translation of the protein and secretion into the supernatant of the host cells.

A variety of secretion signals are also available for expression in yeast host cells, for example S. cerevisiae. These include Prepro alpha factor, HSp150, PHO1, SUC2, KILM1 (killer toxin type 1), and GGP1.

A cloning site is a sequence that facilitates the cloning of a gene encoding a protein of interest into the expression system. It contains restriction sites, or restriction recognition sites, that is, locations in a DNA molecule that contain specific nucleotide sequences, which are recognized by restriction enzymes (see, for example, in the figures). These are generally palindromic sequences (because restriction enzymes usually bind as homodimers) and a particular restriction enzyme can cut the sequence between two nucleotides within its recognition site, or in a proximal location. Cloning sites are well known to those of skill in the art.

More preferably, the nucleotide expression vector further comprises a heterologous DNA sequence encoding a heterologous protein of interest or a heterologous polypeptide inserted into said cloning sites.

The term "heterologous" refers to a combination of non-natural elements. For example, the present application describes "heterologous DNA sequences" encoding "proteins / polypeptides of interest", said DNA sequences not naturally located on or within a host cell chromosomal site that is used for protein expression. .

In the event that a heterologous DNA sequence encoding a heterologous protein or polypeptide of interest is inserted into the disclosed nucleotide vector, it preferably encodes a fusion polypeptide comprising said signal peptide, said MGMT enzyme, mutant or homolog thereof, and said heterologous protein / polypeptide of interest.

In a preferred embodiment, the DNA sequence encoding said MGMT enzyme is located at the 5 'or 3' end of the DNA sequence encoding said heterologous protein of interest, preferably at the 5 'end. Therefore, the MGMT enzyme binds directly or indirectly to the heterologous protein / polypeptide of interest and preferentially locates at the N-terminus of the heterologous protein / polypeptide of interest.

It is particularly preferred that the DNA sequence encoding said MGMT enzyme thereof is located at the 5 'end of the DNA sequence encoding said heterologous protein / polypeptide of interest, in the event that the domain of activity of the heterologous protein / polypeptide of interest is located in its C-terminal part, such as IFNa. In the same way, it could be particularly preferred that the DNA sequence coding for said MGMT enzyme is located at the 3 'end of the DNA sequence coding for said heterologous protein / polypeptide of interest, in the case that the domain of activity of the heterologous protein / polypeptide of interest is located in its N-terminal part.

More precisely, the present application discloses a vector for the expression of recombinant proteins in host cells, preferably in non-vertebrate and / or vertebrate host cells, more preferably in insect cells, comprising a coding nucleotide sequence in a single frame open reading, 5 'to 3' end:

a) a peptide secretion signal that is functional in said host cell,

b) the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63), a mutant or a catalytic domain thereof and

c) a recombinant protein.

In the context of the present application, the term "recombinant protein" or "protein of interest" refers to gene products or polypeptides that are foreign to the protein-producing cell and that are preferably selected from the group consisting of a or more diagnostic and therapeutic proteins or polypeptides.

More preferably, said diagnostic and therapeutic protein or proteins or polypeptide or polypeptides are selected from the group consisting of:

- bacterial or viral immunogenic proteins, more preferably viral proteins (infectious, pathogenic), for example the EDIII protein of Dengue viruses, Japanese encephalitis, tick-borne encephalitis, yellow fever, Usutu, Rocio, de la Murray, Wesselbron, Zika, or West Nile encephalitis, or the nucleoprotein N of Rift Valley fever virus or Tuscany virus, or the soluble form of the E2 coat protein of Chikungunya virus, or the soluble form of the West Nile virus coat protein E, and

- blood factors, anticoagulants, growth factors, hormones, vaccines, therapeutic enzymes, monoclonal antibodies and cytokines (such as IFNa, granzyme M and FasL),

antigens, for example cancer antigens such as testicular cancer antigen SSX2 or the N-terminal region of ERc / mesothelin (NERCMSL),

- antitumor proteins, for example FasL, or heparan sulfate 6-O-endosulfatases (hSULF),

- microbial, viral and / or parasitic polypeptides,

- any other useful proteins (for example contactins).

The FasL protein is a proapoptotic protein that can be used as an antitumor agent. It is encoded, for example, by SEQ ID No. 88.

The hSulf (or hSULF) proteins are heparan sulfate 6-O-endosulphatases that regulate the structure of heparin sulfate and have a drastic impact on the growth and progression of malignant cells in vivo (Dai et al., 2005). In the context of the invention, it is preferably the hSulf2 protein, and more preferably hSulf-2ñTMD, in which the transmembrane domain (TMD) has been removed to increase its solubility, this mutant presenting the amino acid sequence SEQ ID No. 95 and being encoded, for example, in SEQ ID No. 94.

The disclosed vector can also be used to express and purify peptides and / or polypeptides of interest. In the context of the present invention, the terms "peptides" and "polypeptides" are intended to be synonymous, referring to short polymers of amino acid monomers (also referred to as "residues") linked by peptide bonds. Said polymers preferably contain less than 100 residues, more preferably less than 50 residues.

In particular, the disclosed vector can be used to express and purify diagnostic microbial polypeptides, such as bacterial, viral or parasitic polypeptides. Examples of such polypeptides are antigenic peptides, mucins and / or toxins secreted or expressed by bacteria, viruses or parasites. Preferably, said antigenic peptide is expressed by the influenza virus, the hepatitis A virus, the hepatitis B virus, the hepatitis C virus, the hepatitis G virus, the HIV virus, the yellow fever virus , Dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, Usutu or West Nile virus, Rift Valley fever virus or Tuscany virus, Chikungunya virus, syncytial virus respiratory virus, Rocio virus, Murray encephalitis virus, Wesselbron virus, Zika virus, lymphocytic choreomeningitis virus, human paravovirus, human papillomavirus, human cytomegalovirus, or any identified virus. Preferably, said antigenic peptide is expressed by parasitic protozoa (such as Entamoeba histolytica or Giardia lamblia), annelids (such as nematodes, cestodes or trematodes) or arthropods (such as crustaceans, insects or arachnids). Said antigenic peptide is preferably expressed by infectious bacteria, for example of the Streptococcus or Staphylococcus and E. coli genera . Infectious toxins are well known in the art. Examples include botulinum neurotoxins, Clostridium perfringens epsilon toxin , ricin, saxitoxin, shigatoxin, tetrodotoxin, staphylococcal enterotoxins, etc. Mucins are also well known in the art. Examples thereof are MUC5AC, MUC5B and MUC2. Said examples are not limiting and any peptide / polypeptide can be expressed by the present method.

Contactins are a subgroup of molecules belonging to the immunoglobulin superfamily that are exclusively expressed in the nervous system (see the review by Shimoda and Watanabe, 2009). They are involved in psychiatric disorders, particularly autism. The preferred contactins to be produced by the present system are contactins 2 and 4. Contactin 4 (CNTN4) is encoded by, for example, SEQ ID No. 91 (corresponding to amino acids 19 to 990 of the complete protein NP_783200.1 ).

It is known that numerous cancer antigens are efficient vaccine targets for the treatment of cancer. The high quantity production of such polypeptides (see the lists in Cheever et al., 2009) appears to be very important to obtain an efficient cancer vaccine. It is interesting that the disclosed vectors make it possible to obtain a high level of recombinant cancer antigens that can be used in immunotherapy, or to produce antibodies or in cancer diagnostic methods.

SSX2 and NERCMSL are two examples of cancer antigens. The SSX2 cancer antigen is encoded by DNA having SEQ ID No. 76 (GenBank: NM_175698). The N-terminal region of ERC / mesothelin (NERCMSL) is encoded by SEQ ID NO: 83. This antigen is commonly used as a detection antigen in patients suffering from malignant mesothelium.

Any protein can be produced by the present methods.

However, preferred proteins are therapeutic proteins, such as insulin, IFN, FasL, mesothelin, hSULF, or contactins.

More generally, preferred proteins are those that have hitherto been difficult to produce in large quantities. Such proteins are, for example, FasL, granzyme M, hSULF, mesothelin and contactins.

The DNA sequence encoding the fusion polypeptide comprising said signal peptide, said MGMT enzyme, mutant or catalytic domain, and said recombinant protein of interest, can be operatively associated with an inducible promoter that is functional in the same host cells in which it is the peptide signal functional.

More preferably, in the disclosed vector, said open reading frame is operatively associated with an inducible promoter that is functional in the same host cell in which the signal peptide is functional.

A coding sequence is "operatively associated with" an expression control sequence (that is, transcriptional and translational control sequences) in a cell, in the case that the RNA polymerase transcribes the coding sequence into RNA, which is then processed into trans-RNA (in the case of containing introns) and, in the case the sequence encoding a protein, it is translated into that protein.

A "promoter" is a nucleotide sequence from which transcription of operably linked DNA can be initiated downstream (ie, in the 3 'direction in the sense strand of double-stranded DNA). Within the promoter sequence is a transcription start site (conveniently found, for example, by mapping with S1 nuclease), as well as protein binding domains (consensus sequences) responsible for binding of the RNA polymerase.

Promoters that can be used to control gene expression are for example those functional in non-vertebrate cells or in vertebrate cells. For example, for non-vertebrate cells, the regulatory sequences of the metallothionein gene can be used (Brinster et al., Nature 296: 39-42, 1982).

Preferably, the inducible promoter present in the disclosed vector exhibits promoter activity in an insect cell, and more preferably in a Drosophila cell . It is, for example, the Drosophila metallothionein promoter (Lastowski-Perry et al., J. Biol. Chem. 260: 1527, 1985) that drives a high level of gene transcription in the presence of metals, for example CuSO4. Alternatively, the Drosophila 5C actin gene promoter can be used , which is a constitutive promoter and does not require the addition of a metal (BJ Bond et al., Mol. Cell. Biol. 6: 2080, 1986). Examples of other known Drosophila promoters include, for example, the inducible heat shock promoters (Hsp70) and COPIA LTR. The early SV40 promoter provides a lower level of expression than that of Drosophila metallothionein .

Preferably, the inducible promoter present in the disclosed vector exhibits promoter activity in a Drosophila melanogaster cell , preferably in Drosophila S2 cells . It is, for example, the metallothionein promoter that is described in detail in Lastowski-Perry et al., J. Biol. Chem.

260: 1527, 1985.

Suitable promoters for constitutive expression in mammalian cells include the immediate early promoter from cytomegalovirus (CMV), the major late promoter from adenovirus, the phosphoglycerokinase (PGK) promoter, and the thymidine kinase (TK) promoter from herpes simplex virus. (VSH) -1. Inducible eukaryotic promoters regulated by exogenously delivered compounds include, but are not limited to, the zinc-inducible metallothionnein (Mt) promoter, the dexamethasone (Dex) inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter (WO 98/10088), the insect ecdysone promoter, the tetracycline repressible promoter, the tetracycline inducible promoter, the RU486 inducible promoter and the rapamycin inducible promoter.

Preferably, the promoter present in the disclosed vector exhibits promoter activity in a mammalian cell, preferably HeLa cells. It is, for example, the SV 40 promoter.

A range of yeast promoters are available for protein expression in yeast host cells. Some, such as ADH2 and SUC2, are inducible, while others, such as GAPDH, exhibit constitutive expression. Other suitable promoters for yeast expression include the TEF, PGK, MF alpha, Cy C-1, GAL-1, GAL4, GAL10, PHO5, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH) and alcohol dehydrogenase ( ADH).

For use in plant cells, the most commonly used promoter is the Cauliflower Mosaic Virus (CaMV) 35S promoter or its enhanced version, although one of several alternative promoters may be used, such as the 3mas hybrid promoter ( ocs). or the ubiquitin promoter from corn and A. Thaliana. In contrast to such constitutive promoters, the rice RAmy3D α-amylase promoter is induced by sugar deprivation (Hellwig S. et al., 2004).

Promoters suitable for expression in an E. coli host cell include, but are not limited to, the bacteriophage lambda pL promoter, the lac promoter, and the t Rp and IPTG inducible pTAC promoters.

It is preferred that the peptide secretion signal and the inducible promoter are functional in the same host cell.

More preferably, the peptide secretion signal and inducible promoter are functional in both Drosophila S2 cells and vertebrate cells.

The term "inducible" as applied to a promoter is well known to those of skill in the art. In essence, expression under the control of an inducible promoter becomes "activated" or increased in response to the application of a stimulus. The nature of the stimulus varies between promoters. Some inducible promoters cause low or undetectable levels of expression (or no expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus.

After constructing an appropriate vector and transfecting it into the selected host cell, preferably a Drosophila cell line , expression of a heterologous protein is induced by the addition of an appropriate inducing agent to the inducible promoter. For example, cadmium or copper are inducing agents for the Hsp70 promoter. For constitutive promoters, such as the 5C actin promoter, no inducing agent is required for expression.

The human MGMT enzyme of the invention is preferably encoded by the sequence of the reference human MGMT gene NM_002412.3, the gene ID 4255 (SEQ ID No. 3) or by the optimized sequence SEQ ID No. 68 (comprising only 50% G / C). However, any homologous sequence thereof can be used, provided that it encodes a functional MGMT enzyme, mutant or catalytic domain thereof, preferably SEQ ID No. 4 or SEQ ID No. 2. Preferred DNA sequences coding for said MGMT mutant are the SNAP DNA sequences SEQ ID No. 1 or the DNA sequences SEQ ID No. 47 or SEQ ID No. 67 coding for SEQ ID No. 2 but having a G / 51% C.

In another embodiment, the disclosed nucleotide vector encodes at least a fragment of the MGMT enzyme (for example a fragment of SEQ ID No. 4) or a fragment of a homologue thereof (for example a fragment of the MGMT mutant of sequence SEQ ID No. 2) that preserves the biological activity of increasing the expression of the protein of interest by a factor of at least 0.5 times the level obtained with the full-length enzyme of which it is a fragment. By way of example, in the case that the production level is 100 mg / l with the full-length enzyme of SEQ ID No. 4, any fragments of SEQ ID No. 4 that have a production level are included of at least 50 mg / l (under the same experimental conditions as for the full-length enzyme of s Ec ID n ° 4).

In another embodiment, the nucleotide expression vector encodes at least one peptide cleavage site that is preferentially located between the MGMT enzyme or its catalytic domain and the recombinant protein of interest.

A peptide cleavage site (also called a "peptide cleavage site") is an amino acid sequence that is recognized by at least one protease enzyme (eg, a serine protease or a cysteine protease, among others). An example of a peptide cleavage site is the enterokinase cleavage site of SEQ ID No. 62 (AspAspAspAspLys / Asp), for example encoded by the DNA sequence SEQ ID No. 12. Enterokinase is a serine protease enzyme (EC 3.4.21.9) which is known to convert inactive trypsinogen to active trypsin by cutting at the C-terminal end of the sequence: Val - (Asp) 4 - Lys - Ile - Val ~ (trypsinogen) ^ Val - (Asp) 4-Lys (hexapeptide) Ile-Val ~ (trypsin). Enterokinase cuts after lysine in the case that Lys is preceded by four Asps and not followed by a proline residue.

Another useful peptide cleavage site is the cleavage site of the so-called "TEV protease", which has the amino acid sequence SEQ ID No. 53 or SEQ ID No. 65 (Glu Asn Leu Tyr Phe Gln Gly or Ser), and which is, for example, encoded by the DNA sequence SEQ ID No. 52 or SEQ ID No. 66. TEV protease is the common name for the 27 kDa catalytic domain of the nuclear inclusion of a protein encoded by the tobacco etch virus. It is commercially available (Invitrogen).

Dengue virus serotype 1 (SEQ ID NO: 61) membrane precursor prM cleavage site can also be used in the present vector.

In another embodiment, the nucleotide expression vector further encodes a label, preferably located at the C-terminus of the recombinant protein in the fusion polypeptide of the invention (comprising the signal peptide, the MGMT protein or homologue thereof , and the recombinant protein).

In the context of the present application, a "label" is intended to facilitate the recovery of the polypeptide from the crude lysate of host cells and is preferably selected from the group comprising: fluorescent proteins, poly-histidine tags (poly- his) or poly-histidine-glycine (poly-his-gly), influenza HA tags, c-myc tag, herpes simplex virus glycoprotein D (gD) tags, Flag peptides, alpha-tubulin epitopes or tags T7 gene 10 protein peptides. However, any other marking could be useful. In a preferred embodiment, the vectors comprise the DNA encoding a hexa-histidine tag having SEQ ID No. 14.

In another embodiment, the nucleotide expression vector further encodes one or more spacer sequences, preferably located between the MGMT enzyme (or its catalytic domain) and the recombinant protein of interest and / or between the recombinant protein of interest and the label. .

A spacer sequence is an amino acid sequence comprising at least three amino acids, intended to spatially separate two linked polypeptides (subsequently joining these polypeptides in a hint). Said spacer may be, for example, the amino acid sequence Glycine-Glycine-Glycine-Serine (GGGS, SEQ ID No. 63) and the DNA spacer sequence encoding the same may be SEQ ID No. 13. In the context of In the present application, said DNA sequence is hereinafter referred to as "DNA spacer sequence" and is located between the DNA encoding MGMT or the catalytic domain thereof and the recombinant DNA sequence, preferably upstream of the DNA sequence. encoding the peptide cleavage site.

The nucleotide expression vector disclosed in the present application can have the sequence SEQ ID No. 9, the sequence SEQ ID No. 10 or the sequence SEQ ID No. 64 (corresponding to empty vectors without a recombinant gene of interest inserted into cloning sites). In a particular embodiment, the disclosed vector can encode:

- an insect BiP peptide signal (which is preferably functional in Drosophila S2 cells ) or a BiP-like signal as defined above,

- an MGMT protein of SEQ ID No. 4 or a SNAP protein of SEQ ID No. 2,

- a recombinant protein of interest,

- an enterokinase peptide cleavage site or a pro-TEV cleavage site as defined above,

- a poly-histidine label, and

- two spacer sequences having the amino acid sequence Glycine-Glycine-Glycine-Serine (GGGS, SEQ ID No. 63).

In a more preferred embodiment, the disclosed expression vector encodes:

- an insect BiP signal peptide of SEQ ID No. 48,

- an MGMT protein of SEQ ID No. 4 or a SNAP protein of SEQ ID No. 2,

- a recombinant protein of interest,

- an enterokinase peptide cleavage site of SEQ ID No. 62,

- a poly-histidine label, and

- two spacer sequences having the amino acid sequence Glycine-Glycine-Glycine-Serine (GGGS).

In another preferred embodiment, the disclosed expression vector encodes:

- a peptide signal of the BiP type of SEQ ID No. 51,

- an MGMT protein of SEQ ID No. 4 or a SNAP protein of SEQ ID No. 2,

- a recombinant protein of interest,

- a peptide cleavage site of pro-TEV of SEQ ID No. 53,

- a poly-histidine label, and

- two spacer sequences having the amino acid sequence Glycine-Glycine-Glycine-Serine (GGGS).

Said vectors can comprise, for example, the sequence SEQ ID No. 19 (in the case that the protein of interest is the nucleoprotein N of the Rift Valley fever virus (FVR), SEQ ID No. 20 (in the case that the protein of interest is the nucleoprotein N of the West Nile virus), SEQ ID No. 21 or No. 57 or No. 72 or No. 74 (in case the protein of interest is IFNa), SEQ ID No. 77 , No. 79 or 81 (in the case the protein of interest is the SSX2 cancer antigen), SEQ ID No. 55 (in the case the protein of interest is granzyme M), SEQ ID No. 89 (in the case that the protein of interest is FasL), ^s E ^c ID n ° 84 or n ° 86 (in the case that the protein of interest is cancer antigen NERCMSL) or SEQ ID No. 92 (in case the protein of interest is contactin CNTN4).

The present application further discloses a vector for the expression of recombinant proteins in host cells, comprising a coding nucleotide sequence in a single open reading frame, from 5 'to 3':

a) a peptide secretion signal,

b) an MGMT protein of SEQ ID No. 4 or a SNAP protein of SEQ ID No. 2,

c) at least one peptide cleavage site,

d) a poly-histidine label, and

e) at least one spacer sequence.

In a preferred embodiment, said peptide secretion signal is the BiP-type peptide signal of SEQ ID No. 50.

In a more preferred embodiment, said vector comprises two pro-TEV peptide cleavage sites of SEQ ID No. 52 and / or two spacer sequences having the amino acid sequence SEQ ID No. 63.

In a particularly preferred embodiment, said vector comprises the sequence SEQ ID No. 59 or SEQ ID No. 69, referring to said sequences in the present application as the universal DeSNAP cassette "DeSNAP Univ" and the DeMGMT cassette "DeMGMT Univ ", respectively.

These sequences "DeSNAP Univ" (SEQ ID No. 59) and "DeMGMT Univ" (SEQ ID No. 69) are considered "universal" sequences since they can be inserted into any type of vector intended for the transfection of host cells in order to to produce heterologous proteins, that is, vertebrate vectors (such as the pcDNA3 vector or the pCI-neo vector), as well as non-vertebrate vectors (such as pMT / BiP / V5-HisA, which is useful in the DES system; see examples later).

Examples of plasmid comprising said universal sequences are SEQ ID No. 64 (pUC57 comprising DeSNAP Univ) and SEQ ID No. 71 (pUC57 with DeMGMT Univ).

After cloning the heterologous sequence of a protein of interest herein, said vector can advantageously be transfected into vertebrate or non-vertebrate host cells, so that the protein of interest is produced in high amounts.

The present application also discloses the recombinant cell, which is stably transfected by said DeSNAP Univ or DeMGMT Univ vector, that is, by the expression vector comprising a coding nucleotide sequence in a single open reading frame, from 5 'to 3 ':

a) a peptide secretion signal,

b) an MGMT protein of SEQ ID No. 4 or a SNAP protein of SEQ ID No. 2,

c) at least one peptide cleavage site,

d) a poly-histidine label, and

e) at least one spacer sequence,

each component being as defined above.

It preferably comprises the plasmids of SEQ ID No. 64 (pUC57 comprising DeSNAP Univ) or SEQ ID No. 71 (pUC57 with DeMGMT Univ) or at least the nucleotide sequence SEQ ID No. 59 (DeSNAP Univ) or SEQ ID No. 69 (DeMGMT Univ).

Preferably, said recombinant cell is an E. coli cell .

Said recombinant cell is used in order to amplify and purify the expression vectors, preferably those that comprise DeSNAP Univ of SEQ ID No. 59 (such as SEQ ID No. 64) or DeMGMT Univ of SEQ ID No. 69 (such as SEQ ID No. 71).

Therefore, the present application also discloses the use of said recombinant cell for the production of any expression vector defined above.

Nucleotide expression vectors may further comprise a gene encoding a selection marker and / or a terminator sequence.

Selection marker genes that can be included in the construct are typically those that confer selectable phenotypes, such as antibiotic resistance (e.g. blasticidin, ampicillin, kanamycin, hygromycin, puromycin and chloramphenicol).

The present application also discloses a fusion polypeptide comprising a peptide secretion signal that is functional in host cells, preferably in non-vertebrate or vertebrate cells, more preferably in insect cells, and the enzyme 6-methylguanine-DNA-methyltransferase ( MGMT) (EC 2.1.1.63), mutant or catalytic domain thereof as defined above.

In this fusion polypeptide of the disclosure, said MGMT enzyme is preferably the protein of SEQ ID No. 4, the SNAP protein mutant of SEQ ID No. 2, or a homologue thereof.

This fusion polypeptide further comprises a recombinant protein of interest as defined above, preferably located at the C-terminal end of the MGMT enzyme or catalytic domain thereof, and / or a label, as defined above. Said label is preferably a polyhistidine label and is preferably located at the C-terminus of the recombinant protein of interest.

Said fusion polypeptide can be the amino acid sequence of SEQ ID No. 33 to No. 43, SEQ ID No. 56 or SEQ ID No. 58 (in the case where the recombinant protein of interest is GrM), SEQ ID No. 73 or 75 (in case the recombinant protein of interest is IFNa), SEQ ID No. 78 or 80 or 82 (in case the recombinant protein of interest is the SSX2 cancer antigen), SEQ ID No. 85 or No. 87 (in the case that the recombinant protein of interest is NERCMSL), SEQ ID No. 90 (in the case that the recombinant protein of interest is FasL) or SEQ ID No. 93 (in the case the recombinant protein of interest is CNTN4).

Interestingly, such fusion proteins can be stored at 4 ° C for several months without degradation. This in vitro stabilization effect during storage could be the result of the scaffolding properties of the MGMT protein and / or of the high concentration obtained thanks to the presence of the MGMT protein (typically at least 40 mg / ml ).

More importantly, the association with MGMT stabilizes the recombinant proteins during the purification procedure of the secreted proteins. In this way, it could be used to stabilize recombinant proteins in vivo after administration to a subject in need. Coupling with MGMt would be a means to increase the life span of said recombinant proteins in vivo. This in vivo stabilization effect is currently being investigated.

The present application also discloses a recombinant non-vertebrate host cell comprising the expression vector described above.

The non-vertebrate cells can be any cells from the Insect, Arachnid, Crustacean, Mollusc, Annelid, Cirripedian, Radiate, Celenterate and Infusoria divisions. In the context of the present application, the non-vertebrate cells are preferably insect cells, such as Drosophila or Mosquito cells . Most preferably they are Drosophila S2 cells .

Drosophila S2 cells have been widely described. They are especially suitable for high-yield protein production because they can be maintained in suspension cultures at room temperature (24 ± 1 ° C). The culture medium was M3 supplemented with 5% to 10% (v / v) of heat inactivated fetal calf serum (FBS). In a preferred embodiment, the culture medium contains 5% FBS. After induction, cells are cultured in serum-free medium. In such medium, the S2 cells can be cultured in suspension cultures, for example in shake flasks of 250 to 2,000 ml under shaking at 50-60 rpm. Cell densities typically remain between 106 and 107 cells per ml.

The present application also discloses the recombinant Drosophila S2 cells comprising the present expression vectors, said expression vectors preferably comprising the nucleotide sequence selected from the group consisting of:

- the plasmid sequence SEQ ID n ° 64 (pUC57 with DeSNAP Univ) or the nucleotide sequence cloned in the cell that has been deposited according to the Budapest Treaty at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25 , rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I-4581,

- the vector comprising SEQ ID No. 19 or the nucleotide sequence cloned in the cell that has been deposited according to the Budapest Treaty at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on August 19, 2010, with number CNCM I-4357,

- a vector comprising SEQ ID No. 22 or the nucleotide sequence cloned in the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, on 27 October 2010 with the number CNCM I-4381,

- a vector comprising SEQ ID No. 21 or the nucleotide sequence cloned in the cell that has been deposited at the Center National de Culture et de Microorganismos (CNCM), Institut Pasteur, on October 27, 2010 with the number CNCM I -4382,

- the vector of SEQ ID No. 9 or the nucleotide sequence cloned in the cell that has been deposited at the Center National de Culture et de Microorganismos (CNCM), Institut Pasteur, on September 29, 2010 with the number CNCM I- 4368, and

- a vector comprising SEQ ID No. 20 the nucleotide sequence cloned in the cell that has been deposited at the Center National de Culture et de Microorganismos (CNCM), Institut Pasteur, on September 29, 2010 with the number CNCM I -4369,

- the vector of SEQ ID No. 71,

- a vector comprising SEQ ID No. 57 or No. 72 or No. 74 (in which the protein of interest is IFNa), SEQ ID No. 77, No. 79 or No. 81 (in the event that the protein of interest is the cancer antigen SSX2), SEQ ID No. 55 (in case the protein of interest is granzyme M), SEQ ID No. 89 (in case the protein of interest is FasL), SEQ ID n ° 84 or 86 (in case the protein of interest is the cancer antigen NERCMSL) or SEQ ID n ° 92 (in the case that the protein of interest is contactin CNTN4) or SEQ ID n ° 96 (in case the protein of interest is hSULF-2ñTMD).

Such stably transfected S2 cells can also be selected from the group consisting of:

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on August 19, 2010, with the number CNCM I- 4357,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on October 27, 2010, with the number CNCM I- 4381,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on October 27, 2010, with the number CNCM I- 4382,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on September 29, 2010, with the number CNCM I- 4368,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on September 29, 2010, with the number CNCM I- 4369,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4565,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4566,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4567,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4568,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4569,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4570,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4571,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 5, 2011, with the number CNCM I- 4572,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 8, 2011, with the number CNCM I- 4576,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 8, 2011, with the number CNCM I- 4577,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 8, 2011, with the number CNCM I- 4578,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 8, 2011, with the number CNCM I- 4579,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 8, 2011, with the number CNCM I- 4580,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I- 4582,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I- 4583,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I- 4584,

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I- 4585, and

- the cell that has been deposited at the Center National de Culture et de Microorganismes (CNCM), Institut Pasteur, 25, rue du Docteur Roux, 75724 Paris Cedex 15, France, on December 9, 2011, with the number CNCM I- 4586.

The recombinant cell deposited with the number CNCM I-4357 is the stable S2 cell line of Drosophila macrophages comprising the plasmid vector SEQ ID No. 19 (pMT / BiP / SNAP-FVR.N / His ^tag ), in which FVR.N is the Rift Valley fever virus (RVF) N antigen (see Brehin et al., Virology 371: 185, 2008).

The recombinant cell deposited with the number CNCM I-4381 is the stable S2 cell line of Drosophila macrophages that comprises a plasmid vector pMT / BiPN5-His ^tag in which SEQ ID No. 22 (SNAP / WN.EDIII) has been inserted after the BiP sequence, where NO.EDIII is domain III of West Nile virus glycoprotein E.

The recombinant cell deposited with the number CNCM I-4382 is the stable S2 cell line of Drosophila macrophages that comprises the plasmid vector pMT / V5-His ^tag in which SEQ ID No. 21 (BiP / SNAP / IFNa1 has been inserted ). IFNa1 is human interferon alpha 1 of SEQ ID No. 32 (Mokkim et al., Protein expression purif.

63: 140, 2009).

The recombinant cell deposited with the number CNCM I-4369 is the stable S2 cell line of Drosophila macrophages that comprises a plasmid vector pMT / BiPN5-His ^tag in which SEQ ID No. 20 (NO.sE/SNAP/His ^tag ), where NO.sE is the soluble form of West Nile virus coat protein E.

The recombinant cell deposited with the number CNCM I-4369 is the stable S2 macrophage cell line comprising a plasmid vector pMT / BiPN5-His ^tag containing SEQ ID No. 20 (NO.sE/SNAP/His ^tag ), in the that NO.sE is the soluble form of West Nile virus coat protein E.

The recombinant cell deposited with the number CNCM I-4565 is the stable S2 cell line of Drosophila macrophages that comprises a plasmid vector pMT / BiP / SNAP + DVI.EDIII / His ^tag in which DV1.EDIII encodes the EDIII protein of the virus Dengue 1 and presents the sequence SEQ ID No. 27.

The recombinant cell deposited with the number CNCM I-4566 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + DV2.EDNI / His ^tag in which DV2.EDIII encodes the virus EDIII protein Dengue 2 and presents the sequence SEQ ID No. 28.

The recombinant cell deposited with the number CNCM I-4567 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + DV3.EDIII / His ^tag in which DV3.EDIII encodes the virus EDIII protein Dengue 3 and presents the sequence SEQ ID No. 29.

The recombinant cell deposited with the number CNCM I-4568 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + DV4.EDIII / His ^tag in which DV4.EDIII encodes the EDIII protein of the virus Dengue 4 and presents the sequence SEQ ID No. 30.

The recombinant cell deposited with the number CNCM I-4569 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + YF.EDIII / His ^tag in which YF.EDIII encodes the EDIII protein of the virus of yellow fever and presents the sequence SEQ ID No. 31.

The recombinant cell deposited with the number CNCM I-4570 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + JE.EDIII / His ^tag in which JE.EDIII encodes the EDIII protein of the virus of Japanese encephalitis and presents the sequence SEQ ID No. 25.

The recombinant cell deposited with the number CNCM I-4571 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + USU.EDIII / His ^tag in which USU.EDIII encodes the EDIII protein of the virus Usutu and presents the sequence SEQ ID No. 24.

The recombinant cell deposited with the number CNCM I-4572 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + TBE.EDIII / His ^tag in which TBE.EDIII encodes the EDIII protein of the virus of tick-borne encephalitis and presents the sequence SEQ ID No. 26. The recombinant cell deposited with the number CNCM I-4576 is the stable S2 cell line of Drosophila macrophages that comprises a plasmid vector pMT / BiP / SNAP + MVE. EDIII / His ^tag in which EVM.EDIII encodes the EDIII protein of Murray Valley encephalitis virus.

The recombinant cell deposited with the number CNCM I-4577 is the stable S2 cell line of Drosophila macrophages that comprises a plasmid vector pMT / BiP / SNAP + Rocio.EDIII / His ^tag in which Rocio.EDIII encodes the EDIII protein of the virus Dew.

The recombinant cell deposited with the number CNCM I-4578 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + SLE.EDIII / His ^tag in which ESL.EDIII encodes the EDIII protein of the virus of Saint Louis encephalitis.

The recombinant cell deposited with the number CNCM I-4579 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + WSL.EDIII / His ^tag in which WSL.EDIII encodes the EDIII protein of the virus Wesselbron.

The recombinant cell deposited with the number CNCM I-4580 is the stable S2 cell line of Drosophila macrophages which comprises a plasmid vector pMT / BiP / SNAP + Zika.EDIII / His ^tag in which Zika.EDIII encodes the EDIII protein of the virus Zika.

The recombinant cell deposited with the number CNCM I-4583 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + SSX2 / His ^tag in which SSX2 is SEQ ID No. 76.

The recombinant cell deposited with the number CNCM I-4584 is the Drosophila macrophage stable S2 cell line comprising a plasmid vector pMT / BiP / SNAP + NERCMSL / His ^tag in which NERCMSL is SEQ ID No. 83.

The recombinant cell deposited with the number CNCM I-4585 is the stable S2 cell line of Drosophila macrophages comprising a plasmid vector pMT / BiP / SNAP + GrM / His ^tag in which GrM is SEQ ID No. 54.

The recombinant cell deposited with the number CNCM I-4586 is the stable Drosophila macrophage cell line S2 comprising a plasmid vector pMT / BiP / ProTEV / His ^tag in which proTEV is SEQ ID No. 52.

The present application also discloses a vertebrate recombinant host cell that is stably transfected with the present expression vector.

Preferably, said recombinant vertebrate cell is a mammalian cell, preferably a CHO, YB2 / O, COS, HEK, NIH3T3, HeLa cell or derivatives thereof. More preferably, in this case, the disclosed expression vector comprises SEQ ID No. 57 or No. 72 or No. 74 (in case the protein of interest is IFNa), SEQ ID No. 77, No. 79 or 81 (in the case that the protein of interest is the SSX2 cancer antigen), SEQ ID No. 55 (in the case that the protein of interest is granzyme M), SEQ ID No. 89 (in the case of that the protein of interest is FasL), SEC ⁱ D No 84 on 86 (in the case the protein of interest is the antigen the NERCMSL cancer) or SEQ ID No. 92 (in the case of the protein interest is contactin CNTN4) or SEQ ID No. 96 (in the case that the protein of interest is hSULF-2 ^ñTMD ).

The present application discloses a method for increasing the expression of the recombinant protein or proteins that comprises co-expressing said protein or proteins with a peptide secretion signal, together with the enzyme 6-methylguanine-DNA-methyltransferase (MGMT) (EC 2.1.1.63 ), a mutant or a catalytic domain thereof. Said co-expression is preferably carried out in non-vertebrate cells and, more preferably, insect cells.

More preferably, in said method, the MGMT enzyme is the protein of SEQ ID No. 4 or a homologue thereof, for example the SNAP protein of SEQ ID No. 2 or a homologue thereof.

In the context of the present application, the term "which enhances the expression" of a heterologous protein refers to that the expression of said protein in the supernatant of the recombinant cells or within the cells themselves is improved by a factor of at least 2 times, preferably 5 times, more preferably 10 times, and still more preferably 20 times, compared to the expression and / or secretion of said protein obtained with a recombinant vector of the prior art, that is, it does not co-expresses the protein with a MGMT protein or a SNAP protein. In a preferred embodiment, the term "that enhances expression" refers to the fact that it is possible to recover from the supernatant of host cells that have been transfected with a vector at least 40 mg / l, preferably at least 50 mg / l l, more preferably at least 60 mg / l of a protein of interest.

The term "co-expressing agents" refers to the fact that the DNA sequences encoding i) the recombinant protein, ii) the MGMT enzyme, or mutant or catalytic domain thereof, and iii) the peptide secretion signal, are operably linked and regulated. by the same expression control sequence (ie, transcriptional and translational control sequences). Translation of the DNA sequences encoding the peptide secretion signal, the heterologous protein of interest, and the MGMT enzyme therefore leads to the formation of a fusion polypeptide, in which the proteins can be separated with a spacer sequence and / or or an enzyme cleavage site as defined above.

The "peptide secretion signal" of said fusion polypeptide is a secretion signal that is preferably functional in non-vertebrate cells or in vertebrate cells, or both, and more preferably in insect cells, still more preferably in S2 cells of Drosophila.

Examples of peptide secretion signals that are functional in insect cells are: insect ssBiP of SEQ ID No. 48, the BiP-like signal of SEQ ID No. 51, and any peptide signal present in an arbovirus, for example the protein E for West Nile virus coat (SEQ ID No. 15).

An example of a peptide secretion signal that is functional in both vertebrate and non-vertebrate cells is the BiP-like signal of SEQ ID NO: 51.

The present application discloses a method to improve the production of a recombinant protein of interest or to produce the recombinant proteins in cell culture, comprising the use of the vector as indicated above, or the recombinant host cells as indicated above.

More precisely, said method to improve the production of a recombinant protein of interest or to Producing recombinant proteins in cell culture comprises the steps of:

a) providing a nucleotide expression vector, encoding said protein of interest, b) introducing said expression vector into host cells, preferably non-vertebrate or vertebrate cells,

c) providing expression of the introduced nucleotide in said host cells to produce said recombinant protein of interest.

Preferably, said non-vertebrate host cells are insect cells, for example Drosophila S2 cells .

Preferably, said vertebrate host cells are mammalian cells, for example CHO, YB2 / O, COS, HEK, NIH3T3, HeLa cells or derivatives thereof.

Using said method, the recombinant protein of interest is expressed at a rate of at least 40 mg / ml of the recovered cell culture supernatant or higher.

The use of the Drosophila S2 cell line that secretes the gene product directly to the medium is a preferred embodiment (direct secretion to the medium allows the use of an efficient one-step purification system).

The present application discloses the use of the enzyme 6-methylguanine-DNA-methyltransferase (MGMT) (EC 2.1.1.63), mutant or catalytic domain thereof, to enhance the level of production of one or more recombinant proteins, preferably in host cells of non-vertebrate and / or vertebrate, more preferably in insect cells or mammalian cells, infected with replicative or defective vectors. The MGMT enzyme can be the human MGMT (reference NP_8002403.2) of sequence SEQ ID No. 4, the mouse MGMT identified as NP_032624.1 (SEQ ID No. 45), the rat MGMT identified as NP_036993.1 ( SEQ ID NO: 46), a homologous sequence thereof, or sub-fragments thereof.

Preferably, the mutant MGMT enzyme is the SNAP protein of SEQ ID No. 2 or is homologous thereto, that is, it is at least identical to a level greater than 80%, preferably 85%, more preferably 90% relative to the SNAP protein of sequence SEQ ID No. 2.

Said non-vertebrate host cells are preferably insect cells, for example Drosophila S2 cells.

Preferably, the present application discloses the use of the enzyme 6-methylguanine-DNA-methyltransferase (MGMT) (EC 2.1.1.63), mutant or catalytic domain thereof, to enhance the level of production of one or more recombinant proteins in vertebrate cells , for example in mammalian cells, infected with replicative or defective vectors.

Said vertebrate cells are preferably EBX, CHO, YB2 / 0, COS, HEK, NIH3T3 cells or derivatives thereof.

Furthermore, the present application discloses the use of a DNA sequence encoding an MGMT enzyme, mutant or catalytic domain thereof, to improve the level of production of one or more proteins of interest in recombinant cells.

The present application discloses the use of a DNA sequence encoding an MGMT enzyme, mutant or catalytic domain thereof, to i) stabilize one or more recombinant proteins of interest in vitro and in vivo, and, in this way, ii) enhance the life span in vitro and in vivo.

Said DNA sequence is, for example, the sequence of the human MGMT gene NM_002412.3, gene ID 4255 (SEQ ID No. 3) or any homologous sequence of the same encoding a functional MGMT enzyme, a mutant or a catalytic domain thereof (preferably SEQ ID No. 1, SEQ ID No. 47, SEQ ID No. 67 or SEQ ID No. 68). In particular, the present application discloses the use of the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63), mutants or catalytic domain thereof as a protective polypeptide fused or linked to recombinant proteins to improve the half-life of the recombinant protein in storage medium, in plasma or in buffer, to improve the half-life of the recombinant protein used as medicine or vaccine. This enzyme can be used particularly to improve the half-life of the recombinant protein used in diagnostic kits.

In the context of the present application, the expression "enhance the level of production" or "enhance the level of production" of a heterologous protein refers to the fact that the expression of said protein in the supernatant of said cells or within cells improves by a factor of at least 2 times, preferably 5 times, more preferably 10 times, and still more preferably 20 times, compared to the expression and / or secretion of said protein obtained with a recombinant vector of the art above, that is, it does not comprise the present vector. In a preferred embodiment, the expression "which improves production" refers to the fact that it is possible to recover from the supernatant of the host cells that have been transfected with the present vector at least 40 mg / l, preferably at least 50 mg / l, more preferably at least 60 mg / l of a protein of interest.

In a preferred embodiment, said recombinant protein is selected from: insulin, IFN, SSX2, granzyme M, FasL, mesothelin (NERMCSL), endosulfatase (hSUFL) or contactins.

In a particular embodiment, the present application discloses a method for the production of a recombinant protein of interest, the method comprising the steps of:

(a) obtain a heterologous DNA sequence encoding a recombinant protein of interest,

(b) inserting said heterologous DNA sequence into a nucleotide expression vector, said vector having, for example, the DNA sequence SEQ ID No. 9, SEQ ID No. 10, SEQ ID No. 64 or Ec ID No. 71,

(c) transfecting a host cell (preferably an insect cell or a mammalian cell) with the polynucleotide obtained in step (b),

(d) providing the expression of said polynucleotide obtained in step (c) to produce the protein of interest,

(e) optionally cutting the MGMT polypeptide,

(f) recover the protein of interest,

(g) optionally, recovering the protein of interest.

To carry out the different steps of said method, conventional techniques of molecular biology, microbiology and recombinant DNA can be used, understood within the knowledge of the person skilled in the art. These techniques are explained in detail in the literature. See, for example, Sambrook, Fitsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (DN Glover ed. 1985); Oligonucleotide Synthesis (MJ Gait ed. 1984); Nucleic Acid Hybridization (BD Hames & SJ Higgins, eds. 1984); Animal Cell Culture (RI Freshney, ed. 1986); Immobilized Cells and Enzymes (IRL Press, 1986); BE Perbal, A Practical Guide to Molecular Cloning, 1984; FM Ausubel et al. (editors), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., 1994.

The term "transfection" refers to the introduction of a foreign nucleic acid into a eukaryotic host cell such that the host cell expresses the introduced gene or sequence to produce a desired substance, herein a protein encoded by the gene or sequence. inserted. A host cell that receives and expresses the introduced DNA or RNA has been "transfected" and is a "transfectant" or a "clone". DNA or RNA introduced into a host cell can be derived from any source, including cells of the same genus or species as the host cell or cells of a different genus or species.

The transfection of the host cells with the polynucleotides can be carried out by a conventional method of the art, for example by transfection, infection or electroporation. In another embodiment, the vector can be introduced in vivo by lipofection (in the form of naked DNA) or with other transfection facilitating agents (peptides, polymers, etc.). Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7417, 1987). Useful lipid compounds and compositions for nucleic acid transfer are described in WO 95/18863 and W or 96/17823 and in US Patent No. 5,459,127. Lipids can be chemically coupled to other molecules for the purpose of targeted delivery (see Mackey et al., Proc. Natl. Acad. Sci. USA 85: 8027-8031, 1988). Targeted peptides, such as hormones or neurotransmitters, and proteins such as antibodies or non-peptide molecules can be chemically coupled to liposomes. Other molecules are also useful to facilitate the transfection of a nucleic acid in vivo, such as cationic oligopeptides (see WO 95/21931), peptides derived from DNA-binding proteins (see WO 96 / 25508) or cationic polymers (see WO 95/21931). It is also possible to introduce the vector in vivo in the form of a naked DNA plasmid. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, such as electroporation, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter (see Wu et al., J. Biol. Chem. 267: 963-967, 1992; Wu and Wu, J. Biol. Chem.

263: 14621-14624, 1988; Williams et al., Proc. Natl. Acad. Sci. USA 88: 2726-2730, 1991).

The term "providing the expression" of a polynucleotide herein refers to the stimulus of the regulatory sequences present in the vector (for example the stimulus that activates the inducible promoter) and all the necessary components are present in quantity. sufficient for translation of the polynucleotide to occur.

If necessary, cleavage of the MGMT / SNAP polypeptide from the produced fusion protein is obtained by adding a protease having a defined cleavage site in the supernatant or within the recombinant cells. For example, cleavage of the DDDK / D enterokinase cleavage site is obtained by adding an enterokinase enzyme to the supernatant of recombinant cells. Alternatively, the MGMT / SNAP polypeptide can be maintained in a manner that increases the life span of the recombinant proteins.

Furthermore, one of skill in the art will appreciate that an expressed or secreted protein or polypeptide can be detected in the culture medium used to maintain or cultivate the host cells present. The culture medium can be separated from the host cells by known procedures, such as centrifugation or filtration. The protein or polypeptide can then be detected in the cell-free culture medium by taking advantage of the known characteristic properties of the protein or polypeptide. Such properties may include the distinct immunological, enzymatic, or physical properties of the protein or polypeptide. For example, in the event that the protein or polypeptide exhibits unique enzymatic activity, an assay for such activity can be carried out in the culture medium used by the host cells. Furthermore, where antibodies reactive with a given protein or polypeptide are available, such antibodies can be used to detect the protein or polypeptide in any known immunological assay (for example such as in Harlowe et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988).

Recovery of the protein of interest is mediated by means well known in the art, including, but not limited to, preparative gel disk electrophoresis, isoelectric focusing, HPLC, reverse phase HPLC, gel filtration, ion exchange and partition chromatography, precipitation and saline chromatography, countercurrent extraction and distribution, and the like. Because it is preferable to produce the protein of interest in a recombinant system bound to a label, such a label facilitates the recovery of the polypeptide from the crude lysate of the host cell by chromatography on an appropriate solid phase matrix. Alternatively, antibodies raised against the protein or against peptides derived therefrom can be used as recovery reagents.

An additional purification step (g) can be carried out although it is interesting that it is not necessary.

A purified material may contain less than about 50%, preferably less than about 75%, and still more preferably less than about 90% of the cellular components with which it was originally associated. The term "substantially pure" indicates the highest degree of purity that can be achieved using standard purification techniques known in the art.

In one embodiment, the present methods allow obtaining at least 40 mg / l, preferably at least 50 mg / l, more preferably at least 60 mg / l of the substantially pure protein of interest in the cell culture supernatant recovered.

The recombinant proteins of interest and fusion proteins of the invention (ie, recombinant proteins coupled with the MGMT / SNAP polypeptide, which are more stable than recombinant proteins alone) may be useful in a variety of products. For example, such recombinant and / or fusion proteins can be used in pharmaceutical compositions, for example for the treatment of viral infections.

In a preferred embodiment, said recombinant protein is selected from: insulin, IFN, FasL, granzyme M, SSX2, mesothelin (NERMCSL), endosulfatase (hSUFL) or contactins.

In another embodiment, the present application discloses infectious viral particles that comprise the nucleic acid vectors indicated above: Typically, said viral particles are produced by a process that comprises the steps of:

(a) introduce the present viral vector into a suitable cell line,

(b) cultivating said cell line under suitable conditions so as to allow the production of said infectious virus particle,

(c) recovering the infectious virus particle produced from the culture of said cell line, and

(d) optionally purifying said recovered infectious virus particle.

In the event that the viral vector is defective, the infectious particles are usually produced in a complementary cell line or by using a helper virus, which delivers non-functional viral genes in trans . For example, cell lines suitable for complementing adenoviral E1 deletion vectors include 293 cells, as well as PER-C6 cells. Infectious virus particles can be recovered from the culture supernatant or from cells after lysis. They can be further purified according to standard techniques (chromatography, ultracentrifugation on a cesium chloride gradient as indicated in, for example, WO96 / 27677, WO98 / 00524, WO98 / 22588, WO98 / 26048, No. WO00 / 40702, No. EP 1016700 and No. WO00 / 50573).

Thus, the present application also discloses pharmaceutical compositions comprising the expression vector, the recombinant proteins, the fusion proteins, the host cells or the viral particles described above, or any combination thereof. Said pharmaceutical compositions comprise a therapeutic amount of the vector, particles, cells or proteins obtained by the present method in admixture with a pharmaceutically acceptable carrier.

The composition can be routinely administered parenterally, intravenously, or subcutaneously. When administered routinely, the therapeutic composition is in the form of a parenterally acceptable pyrogen-free protein solution. The preparation of such a parenterally acceptable protein solution, with due consideration of pH, isotonicity, stability, and the like, is within the skill of the person skilled in the art.

The administration regimen will be determined by the responsible physician based on the consideration of various factors that modify the action of the drugs, for example, the condition, body weight and diet of the patient, the severity of the infection, the time of administration and other clinical factors. The pharmaceutical carrier and other components of a pharmaceutical composition are selected by the person skilled in the art.

Additionally, the disclosed recombinant and fusion proteins can be used as vaccine components for inoculation of human subjects against viral infection, for example. These proteins can be used alone or with other recombinant proteins or therapeutic vaccine agents. The components of said vaccine will be determined by a person skilled in the art.

The present application further discloses the use of fusion proteins, expression vectors, infectious viral particles, host cells or pharmaceutical compositions described above for the preparation of a medicine, in particular a vaccine.

The present application discloses a method for the treatment or prevention of a human or animal organism, which comprises administering to said organism a therapeutically effective amount of fusion proteins, fusion vectors, infectious viral particles, host cells or cells. compositions described above.

Finally, the proteins, and especially the SNAP-protein fusion of interest, can be useful as diagnostic agents for the detection of the presence of cancer, viral infection or antibodies against viral proteins in biological fluids, such as blood, serum, saliva and Similar. These proteins can also be used in methods to identify and / or isolate viral proteins in biological fluids and tissues. In this way, proteins can be components in kits to carry out these methods.

Thus, in a first aspect, the present invention also relates to the use of recombinant proteins or recombinant proteins tagged with MGMT or SNAP of pathogenic or non-pathogenic microorganisms obtained by any method described above for the identification of the presence of said microorganisms. pathogenic or non-pathogenic in a biological sample. In this regard, said pathogenic microorganism is a virus, and the MGMT or SNAP-tagged protein is a viral protein, such as EDIII of Chikungunya, Dengue, Japanese encephalitis (JE), tick-borne encephalitis (TSG) viruses. ), yellow fever (FA), Usutu (USU) or West Nile, or nucleoprotein N from the Rift Valley fever viruses or the Tuscany virus.

In the context of the invention, said biological sample refers to a blood sample, a urine sample or any biological sample that is possibly infected by viruses.

In this regard, the MGMT protein is the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63) or a homologue thereof, whose sequence has an identity of at least 70% with SEQ ID no: 4, or the SNAP mutant of SEQ ID No: 2 or a homologue thereof, whose sequence has an identity of at least 80% with SEQ ID No: 2.

In this aspect, a fusion polypeptide comprising a viral protein and said MGMT protein is used as a diagnostic agent for the detection of a viral infection in a biological fluid of a patient. Preferably, said viral protein is the EDIII protein from chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), yellow fever (YF), Usutu (USU) or West Nile viruses, or nucleoprotein N from the Rift Valley or Tuscany fever viruses.

Preferably, said biological liquid is urine, blood, serum or saliva.

The present invention relates to a diagnostic immunoassay method using said fusion polypeptide and a biological fluid from a patient.

Preferably, in said diagnostic immunoassay method, said fusion polypeptide is coupled to beads.

More preferably, in said diagnostic immunoassay method, said fusion polypeptide is coupled to beads using a substrate of the MGMT protein as a linker.

This diagnostic immunoassay method can be advantageously used to rapidly and simultaneously detect antibodies in a biological fluid, preferably urine, blood, serum or saliva.

The present application also refers to a diagnostic kit containing the fusion polypeptide defined above, in which said viral protein is a protein from the chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE) viruses , Yellow Fever (YF), Usutu (USU), West Nile, Rift Valley Fever or Tuscany.

This diagnostic kit can be used to identify a viral infection in a biological sample from a patient, such as urine, blood, serum, or saliva.

Examples

1. Construction of one or more plasmids

1.1. The plasmid pMT / BiP / V5-His A was used. It contains 3,642 nucleotides and the following elements:

- metallothionein promoter: bases 412 to 778

- Start of transcription: base 778

- MT forward primer site: bases 814 to 831

- BiP signal sequence: bases 851 to 904 (SEQ ID No. 11).

- Multiple cloning site: bases 906 to 999

- V5 epitope tag: bases 1,003 to 1,044

- polyhistidine region: bases 1,054 to 1,074

- BGH reverse primer site: bases 1,094 to 1,111

- SV40 late polyadenylation signal: bases 1,267 to 1,272

- origin of pUC: bases 1,765 to 2,438 (complementary chain)

- bla promoter : bases 3,444 to 3,542 (complementary strand)

- ORF for ampicillin resistance gene ( bla) bases 2,583 to 3,443 (complementary strand)

The vector pUC57 Amp can also be used for the purposes of the present application. Said vector comprises: - the unique EcoRI cloning site

- the metallothionein promoter,

- the 5 'non-coding region of the genomic RNA of West Nile virus strain IS-98-ST1,

- a translation start codon (ATG),

- the signal peptide of the coat protein E of the West Nile virus strain IS-98-ST1 (SEQ ID No. 15),

- The 3 'non-coding region of the genomic RNA of West Nile virus strain IS-98-ST1 in which they have two repeated sequences and the stem-loop of the 3 'end have been eliminated,

- the SV40 polyA signal motif,

- a single ApaI cloning site .

1.2. SNAP cloning

The amplification of the DNA encoding the sequence SEQ ID No. 2 of the SNAP protein was carried out in the template pMT / BiP / CHIK.sE2 + SNAP tag by PCR using the primer pair 5'-SNAP and 3'-MCS as indicated below.

5'-SNAP primer: 5'-aaaaaagatctgacaaagactgcgaaatg-3 '(SEQ ID No. 7)

3'-MCS primer: 5'-gaggagagggttagggataggcttacc-3 '(SEQ ID No. 8)

The PCR product was then digested with BglII and NotI and inserted between the unique sites BglII (at the 5 'end of MCS) and NotI (at the 3' end of MCS) of the linearized plasmid p / MT / BiP / V5-A in the DES system.

The resulting plasmid is the pMT / BiP / SNAP-Hislabel vector of SEQ ID No. 9, which comprises:

- the sequence of insect BiPmc of SEQ ID No. 11,

- the DNA sequence of SNAP of SEQ ID No. 1,

- the enterokinase cleavage site of SEQ ID No. 12,

- an EcoRV-Smal restriction site ,

- the DNA encoding a His6 tag (SEQ ID No. 14) located downstream of the restriction site, and - two DNA spacer sequences of SEQ ID No. 13 located i) between the enhancer sequence and the EcoRV restriction site -Smal, and ii) between the EcoRV-Smal restriction site and the DNA encoding a His6 tag.

A vector pMT / BiP / SNAP-Hislabel can also be obtained from a backbone of pUC57 and a vector having the sequence SEQ ID No. 10 is obtained. Said vector comprises:

- the unique EcoRl cloning site

- metallothionein promoter

- the 5 'non-coding region of the genomic RNA of West Nile virus strain IS-98-ST1,

- a translation start codon,

- the signal peptide of the coat protein E of the West Nile virus strain IS-98-ST1 of SEQ ID No. 15

- the DNA sequence of SNAP of SEQ ID No. 47,

- unique EcoRV and Smal / Xmal cloning sites for insertion within the framework of the foreign sequence, - the enterokinase cleavage site of SEQ ID NO: 12, located between the SNAP enhancer DNA and the cloning sites,

- a DNA encoding a hexa-histidine tag sequence (SEQ ID No. 14),

- two DNA spacer sequences of SEQ ID No. 13 located i) between the enhancer sequence and the EcoRV-SmaI restriction site, and ii) between the EcoRV-Smal restriction site and the DNA encoding a His6 tag.

- two translation stop codons,

- The 3 'non-coding region of the genomic RNA of the West Nile virus strain IS-98-ST1 in which two repeat sequences and the stem-loop of the 3' end have been deleted,

- polyA signal motifs of S40 and

- a single Apal cloning site .

A pMT / BiP-like / SNAP-His-tag vector can also be obtained from a backbone of pUC57 into which SEQ ID No. 59 (see also Figure 8) has been inserted between the unique EcoRV and HindIII sites. Said vector has the sequence SEQ ID No. 64. It comprises:

- the unique EcoRI cloning site

- metallothionein promoter

- the 5 'non-coding region of the genomic RNA of West Nile virus strain IS-98-ST1,

- a translation start codon,

- The signal peptide of the coat protein E of the West Nile virus strain IS-98-ST1 of SEQ ID No. 15,

- the DNA sequence of SNAP of SEQ ID No. 47,

- unique BamHI, EcoRV, Apal and Xmal cloning sites for in-frame insertion of the foreign sequence,

- two pro-TEV cleavage sites of SEQ ID No. 52, located between the SNAP enhancer DNA and the His tag,

- a DNA encoding a hexa-histidine tag sequence (SEQ ID No. 14),

- two DNA spacer sequences of SEQ ID No. 13 located i) between the enhancer sequence and the EcoRV-Smal restriction site , and ii) between the Apal restriction site and the DNA encoding a Hisa tag.

- two translation stop codons,

- The 3 'non-coding region of the genomic RNA of the West Nile virus strain IS-98-ST1 in which two repeat sequences and the stem-loop of the 3' end have been eliminated,

- polyA signal motifs of S40 and

- Unique ApaI cloning site.

1.3. Cloning a gene of interest

1.3.1. Rift Valley fever virus nucleoprotein N (RVF-N)

Site-directed mutagenesis was carried out on a cDNA encoding the FVR-N protein sequence by PCR using the 5'-N and 3'-N primer pair as listed below.

5'-N primer:

5'- aaaaaggcgcgccagggggtggcggatctgacaactatcaagagcttcgagtccagtttgctgctc- 3 '(SEQ ID No. 17)

3'-N primer:

5'- aaaaaaccggtcaatgatgatgatgatgatgacttccaccgccggctgctgtcttgtaagcctgagcgg- 3 '(SEQ ID No. 18)

1.3.2. Non-viral protein, for example IFNa1 or granzyme M

The human IFNa1 protein sequence of SEQ ID NO: 32 can also be used.

The human granzyme M protein sequence of SEQ ID No. 54 can also be used. Granzyme M is a chymotrypsin-like serine protease that preferentially cleaves its substrates after Met or Leu. It is constitutively expressed in activated innate effector natural killer cells. This protease also exhibits antiviral and antitumor properties (van Domselaar R. et al, The Journal of Immunology, 2010; Hu D. et al., The Journal of Biological Chemistry, 2010).

1.4. Insertion of a protein coding gene in the vector pMT / BiP / SNAP-Hislabel or pMT / type BiP / SNAP-Hislabel, so that the vector pMT / BiP / PROTEIN SNAP-Hislabel or the vector pMT / type BiP / PROTEIN SNAP-Hislabel

1.4.1. FVR.N

The PCR products obtained in section 1.3.1 were digested with BssHIl and AGel and inserted between the unique sites BssHII (at the 3 'end of the SNAP gene) and Agel (at the 3' end of the MCS of the shuttle vector) of the linearized plasmid pMT / BiP / SNAP-Hislabel obtained in section 1.2.

The resulting plasmid is, for example, pMT / BiP / SNAP-FVR.N / His-tag (SEQ ID No. 19).

1.4.2. ED INI proteins from different flaviviruses

To enhance the specificity of recombinant flaviviric antigen-based ELISA or immunoblot assays, protein E antigenic domain III (ED III) appears to be a promising approach (Ludolfs et al., 2007). Recombinant EDIII production of West Nile (NO), Usutu (USU), Japanese encephalitis (JE), tick-borne encephalitis (TSG) viruses, dengue serotypes 1 to 4 (DEN-1, -2 , -3 and -4) and yellow fever (YF) have been tested with the disclosed method.

The NO, USU and EJ viruses belong to the EJ serocomplex. The Drosophila S2 inducible expression system (DES, Invitrogen) has been selected for the mass production of flavivirus individual EDIII in non-vertebrate cells. The synthetic genes encoding the full-length domain III of the E proteins of the flaviviruses NO, USU, EJ, ETG, DEN-1 to DEN-4 and FA are listed in SEQ ID No. 23 to SEQ ID No. 31. The ED III sequences were fused in frame with the C-terminus of the SNAP protein in the pMT / BiP / SNAP-Hislabel plasmid obtained in 1.2 to generate the SNAP-EDIII fusion proteins.

1.4.3. IFNa

The plasmids pMT / BiP / SNAP-IFN-Hislabel (see figure 4) and pMT / type BiP / SNAP-IFN-Hislabel (see data in figure 8) were obtained.

1.4.4. Granzyme M

The plasmid pMT / BiP / SNAP-GrM-Hislabel was obtained (see data in figure 6).

2. Transfection into host cells

2.1. Transfection into S2 cells

The resulting plasmids pMT / BiP / SNAP-His-tagged PROTEIN that allow the production of SNAP-tagged proteins as secreted His-tag-terminated fusion proteins were cotransfected with the selection marker pCo-Blast into S2 cells to generate the stable cell line S2 / sPROTEIN -SNAP-Hislabel showing resistance to ampicillin.

The stable S2 cell lines grown in shake flasks (1000 ml) were stimulated for 10 days with cadmium heavy metal (Cd2 +) and the proteins in the extracellular medium were concentrated and purified.

The accumulation of secreted SNAP-tagged protein was observed in S2 / sPROTEIN-SNAP-Hislabel cell supernatants after 10 days of induction with heavy metal cadmium.

Immunoblotting assays allowed detection of extracellular SNAP-tagged proteins using goat anti-Hislabel serum antibody (see Figures 2B, 3B, 4B and 6C).

2.2. Transfection into HeLa cells

The plasmid pMT / BiP type / SNAP-IFN-His-tag was transfected into HeLa cells.

Immunoblot assays allow detection of SNAP-tagged extracellular IFN using anti-SNAP antibodies (see Figure 7B).

3. Purification of recombinant proteins

Extracellular His-tagged and SNAP-tagged proteins were purified using metal chelating resin and the HPLC method.

3.1. FVR-N and TOS-N

FVR-N

In collaboration with Plate-Forme 5 Production de Protéines recombinantes et d'Anticorps (Institut Pasteur), up to 97 mg of highly purified SNAP-tagged FVR-N protein have been obtained from 2 liters of S2 / sSNAP-FVRV cell supernatants .N-Hislabel 10 days after Cd2 + stimulation.

TOS-N

In collaboration with Plate-Forme 5 Production de Protéines recombinantes et d'Anticorps (Institut Pasteur), up to 41 mg of highly purified SNAP-tagged FVR.N protein have been obtained from 2 liters of S2 / sSNAP-TOS cell supernatants .N-Hislabel 10 days after Cd2 + stimulation.

Summary of production levels:

3.2. Soluble IFNa1

Soluble IFNa1 proteins were released from the SNAP tag by cleavage with the enzyme enterokinase (Novagen kit).

3.3. Antigens of different flaviviruses

SNAP-Tagged Protein Reserves Secreted Using the Drosophila Expression System

• EDIII: antigenic domain III of flavivirus E proteins (Dengue [DEN], West Nile [NO], Japanese encephalitis [EJ], Usutu [USU], tick-borne encephalitis [TSG], yellow fever [FA], encephalitis of the Murray Valley [EVM], Wesselbron [WSL], Rocio, Zika)

• ectoM: ectodomain of Dengue virus type 1 M protein

• RVF N gene: Rift Valley fever virus nucleoprotein N (major viral antigen)

• TOS N gene: Tuscany virus nucleoprotein N (major viral antigen)

• WN sE: soluble form of West Nile virus coat protein E

• CHIK sE2: soluble form of the Chikungunya virus coat protein E2

• SNAP-IFNAI: interferon-alpha 1 in fusion with SNAP.

3.4. Production of granzyme M

10 mg of SNAP-GrM protein was recovered for each liter of culture supernatant in 7 days.

After the purification steps, three forms of SNAP-GrM were detected (see Figure 6C), corresponding to the cleavage of the SNAP protein by the coupled GrM enzyme.

This clearly means that the human protease is active after its production by the disclosed method (see below).

4. Control of SNAP-tagged proteins

Immunoblot assays using specific antibodies (which recognized the protein of interest and / or the histidine tag label) detected substantial production of SNAP-tagged extracellular proteins:

The immunoblot assay detected SNAP-tagged FVR.N extracellular protein using goat anti-Histag serum antibody (Figure 2B). Human and mouse immunological sera against FVR.N specifically recognize SNAP-tagged recombinant FVR.N protein.

Immunoblot assays did not show cross-reactivity between NO ED III and recombinant USUs using specific mouse polyclonal sera despite the high level of sequence similarity. In this way, the soluble SNAP-EDIII protein secreted from the NO, EJ and USU viruses are suitable as recombinant viral antigens for the specific diagnosis of members of the EJ serocomplex since the USU virus and, to a lesser extent, the EJ viruses have been recently identified as potentially emerging arboviruses in Europe.

5. Activity of SNAP-tagged recombinant proteins

• The soluble recombinant SNAP-IFNal protein secreted from induced S2 / SNAP-IFNaI cells shows a potent antiviral effect against CHIKV.

Supernatants (5 ml) of S2 / SNAP-IFNaI cells (5x10 6 cells / ml) stimulated with Cd2 + were collected ten days after induction. Accumulation of soluble SNAP-IFNal protein in cell supernatant was observed by immunoblotting using anti-Hislabel antibody (see below). The antiviral activity of SNAP-IFNaI was evaluated in HeLa cells infected with Chikungunya virus expressing the luciferase gene (Luc). Luc activity was determined 6 h after infection. IFN alfaconl (Infergen) was used as an internal assay to learn of its potent antiviral effect against CHIKV in HeLa cells. The supernatant of S2 / SNAP-Tos.N cells (the Tuscany virus N protein) stimulated with Cd2 + served as a negative control. The graph illustrated in Figure 4C demonstrates that 1 µl of secreted SNAP-IFNaI or 0.1 µg of Infergen could suppress CHIKV replication within infected host cells. The graph shows a dose-dependent effect of SNAP-IFNa. Twenty percent Luc activity was still observed with 0.1 µl of soluble SNAP-IFNaI or 0.01 µg of Infergen. No antiviral effect was observed using SNAP-TOS-N at the highest dose tested.

• Granzyme M is active after being produced in the supernatant of S2 cells

As indicated above, three forms of SNAP-GrM have been detected in the supernatant of S2 cells transfected with the pMT / BiP / SNAP-GrM-Hislabel vector (see Figure 6C).

Said three forms correspond to the cleavage of the SNAP protein by the coupled GrM enzyme. SNAP in effect it contains three potential GrM cleavage sites (see Figure 6B). Immunoassays with anti-His or anti-SNAP antibodies have revealed that these three forms are indeed fragments of the secreted SNAP-GrM fusion protein.

The smallest form (35 kDa) corresponds to GrM, which has been deleted by most of SNAP during the purification procedure.

These results clearly demonstrate that the GrM protease that has been produced by the disclosed system is active, although coupled with the SNAP protein.

This is really interesting because it is known that active human proteases are very difficult to produce recombinantly.

• hSULF-2ñTMD is active after being produced in the supernatant of HEK 293 cells. HSULF-2ñTMD was expressed and purified from HEK-293 cells transfected with a recombinant plasmid pcDNA3 / De SNAPuniv-hSULF-2ñTMD (see figure 13A).

The enzymatic activity of the hSULF-2ñTMD polypeptide obtained in the supernatant was evaluated as follows:

HEK 293 cells were transiently transfected with pcDNA3 / DeSNAP-hSULF2ATMDs. After two days, an aliquot of the cell supernatant was incubated with the non-fluorescent pseudosubstrate 4-methyl-umbelliferone (4-MUS) at a concentration of 20 mM in 50 mM Tris, pH 7.5, with 1220 mM MgC, and incubated ( 1: 1, v / v) with the enzyme (in conditioned medium) for 2 to 4 hours at 37 ° C in a 96-well plate. The enzymatic reaction was stopped by the addition (1: 1 v / v) of NaHCO3 / Na2CO3 1.5 M, pH 10.5 and the generation of the fluorescent product 4-methyl-umbelliferone was monitored by fluorimetry (excitation: 360 nm) . SULF activity values in the cell supernatant were measured from the optical density (OD) at 460 nm.

Interestingly, the secreted protein (SNAP? Coupled) is active, as shown in Figure 13B.

6. Stability of SNAP-tagged recombinant proteins

Unexpectedly, it has been observed that fusion proteins comprising the SNAP peptide are much more stable in vitro than in their absence.

The highly purified CHIK.sE2-SNAP, SNAP-WN.EDIII and SNAP-IFNAI proteins were incubated at the non-saturating concentrations of 0.1 mg / ml (vol .: 0.1 ml) in sterile PBS for 4 days at - 80 ° C, 4 ° C, 25 ° C or 37 ° C, or for two months at the same temperature.

Protein samples (1 µg) were separated on 4-12% SDS-PAGE and visualized with Coomassie G-250 Brilliant Blue pigment using PageBlue protein staining solution (Fermentas).

Figures 10A and B report the results obtained by comparing the stability of three different fusion proteins in vitro.

Importantly, all fusion proteins were apparently intact after two months at 4 ° C and also after four days of incubation at room temperature (25 ° C) or 37 ° C.

In particular, IFN is unaffected after 4 days at body temperature (37 ° C) and is still observed after two months at 37 ° C, so in vivo stability is likely to be highly increased by its coupling with SNAP.

7. In vitro detection of SNAP-FVR.N produced by S2 cells

SNAP-FVR.N fusion proteins that had been produced following the protocols indicated above were used as a diagnostic tool for the detection of anti-RVF.N antibodies in the serum of infected sheep.

Said fusion proteins were tested and compared with commercial detection kits (BDSL RVF IgG Detection Kit and IdVet Multispecies RVF).

The tests were carried out with 46 sheep sera, the sheep being immunized with RVF vaccines. SNAP-FVR.N fusion proteins were used to directly coat the bottom of the wells or were biotinylated and added to streptavidin coated wells.

Anti-RVF antibodies were detected by indirect ELISA method using 96-well microtiter plates directly coated with 0.2 µg of highly purified recombinant SNAP-FVR.N antigen in BS (concentration: 2 µg protein / ml) overnight at 4 ° C. After saturation, the diluted sera were incubated with SNAP-FVR.N. Peroxidase-conjugated goat anti-IgG antibody was used as secondary antibody. ELISA was carried out with the peroxidase substrate system and the optical density (OD) was measured at 450 nm. The sample sera were considered positive if the OD was twice the OD of the non-immunological sera.

Interestingly, the results demonstrate that SNAP-FVR.N fusion proteins provide the same sensitivity and specificity as commercial proteins when used to directly coat wells (not shown). The results are less reproducible in the case that the proteins are biotinylated. The same results were obtained with sera obtained from naturally immunized cattle (data not shown).

These results demonstrate that the fusion proteins of the invention can be used as diagnostic tools for the identification of viral or bacterial infections in biological samples.

8. Multiplex bead-based immunoassay

In the context of the invention, a multiplex bead-based immunoassay was developed for the rapid and simultaneous detection of antibodies against arboviruses in biological fluids.

The system is based on xMAP technology (Luminex Corporation) and uses a mixture of antigen-coated microspheres as capture reagents for specific human immunoglobulins. Different sets of microspheres (Magplex, Luminex Corporation) were coupled with purified MGMT fusion proteins, i.e., SNAP-tagged viral recombinant proteins listed in section 3.3: sSNAP-DVI.EDIII, sSNAP-DV2.EDIII, sSNAP- DV3.EDIII, sSNAP-DV4.EDIII, sSNAP-WN.EDIII, sSNAP-EJ.EDIII, sSNAP-USU.EDIII, sSNAP-ETG.EDIII, sSNAP-FA.EDIII, sSNAP-EVM.EDIII, sSNAP-Rocio. EDIII, sSNAP-WSL.EDIII, sSNAP-ZIKA.EDIII, SNAP-DVlectoM, sSNAP-N.FVR, sSNAP-N.TOS and CHIK.sE2-SNAP. Recombinant antigens were covalently coupled to the carboxyl surface of the microspheres using a substrate of the MGMT protein as a linker (BG-PEG-NH2, New England Biolabs), thus increasing the efficiency of antibody capture compared to the procedures amine coupling standards.

Technical validation using anti-SNAP tag antibodies and specific mouse monoclonal antibodies confirmed the coupling efficiency and demonstrated the long-term stability of the antigens (up to six months). The present application is not limited to viral antigens, since any peptide or polypeptide can be used for the coating of beads and the subsequent capture of antibodies.

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Claims (11)

1. In vitro use of a fusion polypeptide comprising a viral protein and i) the enzyme 6-methylguanine-DNA-methyltransferase (MGMT, EC 2.1.1.63) or a homologue thereof, the sequence of which has an identity of at least 70% with SEQ ID no: 4, or ii) the SNAP mutant of SEQ ID No: 2 or a homologue thereof, whose sequence has an identity of at least 80% with SEQ ID No: 2, as a diagnostic agent for the detection of a viral infection in a biological fluid from a patient. 2. Use according to claim 1, wherein said viral protein is EDIII protein from chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis (TBE), yellow fever (YF), Usutu ( USU) or West Nile, or nucleoprotein N from Rift Valley or Tuscany fever viruses. 3. Use according to claim 1 or 2, wherein said biological fluid is urine, blood, serum or saliva. 4. A diagnostic immunoassay method using the fusion polypeptide as defined in claims 1 to 2 and a biological fluid from a patient. 5. A diagnostic immunoassay method according to claim 4, wherein said fusion polypeptide is coupled to beads. 6. A diagnostic immunoassay method according to claim 4, wherein said fusion polypeptide is coupled to beads using a substrate of the MGMT protein as a linker. 7. A diagnostic immunoassay method according to any one of claims 4 to 6, for rapidly and simultaneously detecting antibodies in a biological liquid. 8. A diagnostic immunoassay method according to claim 7, wherein said biological fluid is urine, blood, serum or saliva. 9. Diagnostic kit containing the fusion polypeptide as defined in claim 1, wherein said viral protein is the EDIII protein from chikungunya, dengue, Japanese encephalitis (JE), tick-borne encephalitis viruses ( TBE), yellow fever (YF), Usutu (USU) or West Nile fever, or nucleoprotein N from Rift Valley or Tuscany fever viruses. 10. Use of the diagnostic kit according to claim 9, to identify a viral infection in a biological sample from a patient. 11. Use according to claim 10, wherein said biological sample is urine, blood, serum or saliva.