The present invention relates to a system for expressing toxic proteins, to an expression vector comprising this system, to a prokaryotic cell transformed with this system, and also to a method for synthesizing a toxic protein using this expression system. The expression system of the invention is characterized in that it comprises successively, in the 5'-3' direction, a nucleotide sequence encoding the Asp-Pro dipeptide and a nucleotide sequence encoding a toxic protein. According to a preferred embodiment of the invention, the expression system also comprises, upstream of the Asp-Pro sequence, a nucleotide sequence encoding a soluble protein. The expression system of the invention makes it possible to construct an expression vector that is useful for transforming a prokaryotic cell such as E. coli, for example in a method for synthesizing the toxic protein.

Description of the Invention

FIELD OF THE INVENTION

The present invention relates to systems for expressing toxic proteins, to expression vectors comprising one of these systems, to prokaryotic cells transformed with these systems, and also to a method for synthesizing a toxic protein using these expression systems.

It enables, for example, the overproduction in a prokaryotic cell, for example Escherichia coli (E. coli), of toxic hydrophobic proteins or peptides, for example the overproduction of transmembrane domains of viral envelope proteins.

It finds many applications in particular in research concerning the mechanisms of viral infections, and in the search for and development of novel active principles for combating viral infections.

In the description which follows, the references between square brackets [ ] refer to the attached reference list.

BACKGROUND OF THE INVENTION

Determination of the three-dimensional (3D) structure is a decisive step in the structural and functional understanding of proteins.

Very great efforts and means have been, and are being, used to achieve this aim, and have been amplified with the accumulation of data provided by the genome sequencing programmes [1].

The two main techniques for establishing these protein structures are X-ray diffraction, carried out using crystallized proteins, and nuclear magnetic resonance (NMR) carried out using proteins in solution. NMR, which is very suitable for studying proteins with a molecular mass of less than 20 kDa, requires however, like X-ray diffraction, the production of large amounts of material. It also means, in most cases, that material enriched in .sup.15N and/or .sup.13C must be prepared.

In this context, the bacterium is a means of production that is widely used by the scientific community [2]. The overexpression of proteins in bacteria does not, however, occur without problems. In fact, it gives rise to three situations: The first case, which is ideal, is that where the protein is overproduced in a form that is correctly spatially folded during its synthesis in vivo. This is not a rare situation, but neither is it frequent. It concerns essentially soluble proteins that are small, i.e. approximately 20 to 50 kDa. The second case, the most common, is that where the protein is overproduced and aggregated in the form of inclusion bodies. This concerns polytopic and/or large proteins. In this case, the kinetics of folding of the protein are clearly slower than its rate of biosynthesis. This promotes exposure of the hydrophobic regions of the protein, that are normally buried in the core thereof, to the aqueous solvent and generates non-specific interactions that result in the formation of insoluble aggregates. According to the degree of disorder of this folding, the inclusion bodies can be solubilized/unfolded under non-native conditions, with urea or guanidine. The solubilized protein is then subjected to various treatments, such as dialysis or dilution, so as to promote, successfully in certain cases, a native 3D folding. The third case is that where the expression engenders a varying degree of toxicity. This goes from an absence of expression product if the bacterium manages to adapt itself, to death of the bacterium if the product is too toxic. It is a case which occurs quite frequently and most commonly with membrane proteins or membrane protein domains, for instance those of the envelope proteins of the hepatitis C virus [5] or of the human immunodeficiency virus [6].

The problem of toxicity relates essentially to the expression of membrane proteins, i.e. proteins having a hydrophobic domain. Now, these proteins are of growing interest. Firstly, they are relatively numerous since the establishment of the various genomes confirms that they represent approximately 30% of the proteins potentially encoded by these genomes [7]. Secondly, they constitute 70% of the therapeutic targets and their alteration is the cause of many genetic diseases [8].

It is therefore essential to develop methods that facilitate or allow the expression of such proteins or of their membrane portion.

Efforts have been made in this respect with, for example, the development of bacterial strains that either show better tolerance to the expression of membrane proteins [9, 10], or have a stricter regulation of the mechanism in the expression, as in the case of the E. coli strain BL21(DE3)pLysS developed by Stratagene. However, these improvements do not make it possible to eliminate the toxicity phenomenon in all cases, in particular in the expression of hydrophobic peptides corresponding to membrane anchors.

The treatment of hepatitis C currently represents one of the major high-stakes areas of medicine. Hepatitis C is caused by the hepatitis C virus (HCV) of the family of flaviviridae and which specifically infects hepatic cells [11]. This virus consists of a positive RNA of approximately 9500 bases which encodes a polyprotein of 3033 residues [13], symbolized in the attached FIG. 1 (see Original Patent) by the rectangle 1A. This polyprotein is cleaved, after expression, by endogenous and exogenous proteases, so as to give rise to 10 different proteins. Two of them, called E1 and E2, are glycosylated and form the envelope of the virus. They each have membrane domains called TM, in particular TME1 for the E1 protein and TME2 for the E2 protein. The cleavage positions that generate them are indicated in FIG. 1 by arrows with, mentioned below, a number which corresponds to the position in the polyprotein of the first amino acid of sequence resulting from the cleavage. The E1 and E2 proteins are symbolized by a rectangle. The white portion of each rectangle corresponds to the ectodomain (ed) and the shaded domain to the transmembrane region (TM). The primary sequence of the TMs is indicated at the bottom of the figure in one-letter-code, with numbers corresponding to the position of the amino acids in the polyprotein located at the ends of these domains. The stars indicate the hydrophobic amino acids. These membrane domains or membrane regions of the virus have particular association properties that condition the structuring of the viral envelope [12]. In this respect, they constitute potential therapeutic targets. An understanding of the mechanism of association of the virus requires studies of the 3D structure of these domains, in particular by means of the abovementioned techniques, which involves producing these peptides in abundant amounts, and also preferably via the biosynthetic pathway in order to allow .sup.15N and/or .sup.13C isotope labelling.

The various E1 expression trials of the prior art, in particular in E. coli [14][5] or in sf9 insect cells infected with baculoviruses [15], have not made it possible to overproduce this E1 protein, in particular due to the toxicity induced by its expression, including in the "resistant" E. coli BL21(DE3)pLysS strains described above. There has been no E2 protein overexpression trial in bacteria. These toxicity problems are essentially due to the C-terminal region of the two proteins, that is rich in hydrophobic amino acids which form transmembrane domains that provide the anchoring to the membrane of the endoplasmic reticulum.

There is therefore a real need for a system for expressing toxic proteins which does not have the drawbacks, and limitations, deficiencies and disadvantages of the techniques of the prior art.

In addition, there is a real need for an expression vector comprising such a system for expressing toxic proteins, making it possible to carry out a method for producing toxic proteins which does not have the drawbacks, limitations, deficiencies and disadvantages of the techniques of the prior art.

SUMMARY OF THE INVENTION

The aim of the present invention is precisely to provide a system for expressing a toxic protein, which satisfies, inter alia, the needs indicated above.

This aim, and others, are achieved, in accordance with the invention, by means of an expression system characterized in that it comprises successively, in the 5'-3' direction, a nucleotide sequence encoding the dipeptide Asp-Pro, referred to below as dp sequence, and a nucleotide sequence (pt) encoding a toxic protein (Pt). This system will be identified below by: dp-pt.

DETAILED DESCRIPTION OF THE INVENTION

According to a particularly preferred embodiment of the present invention, the expression system also comprises, upstream of the dp sequence, a nucleotide sequence (ps) encoding a soluble protein (Ps). This soluble protein may be, for example, glutathione S-transferase (GST) or thioredoxin (TrX) or another equivalent soluble protein. This expression system according to the invention will be identified below by: ps-dp-pt.

The dp-pt expression system of the present invention, which comprises a sequence encoding Asp-Pro (DP in one-letter code) placed upstream of the nucleotide sequence of the toxic protein, makes it possible, entirely unexpectedly, to suppress the toxic effect of the protein for the host cell. In addition, the inventors have noted that, entirely surprisingly, the suppression of toxicity of the protein in the host is even more effective with the ps-dp-pt expression system when the toxic peptide is produced as a C-terminal fusion with a soluble protein, for example glutathione S-transferase or thioredoxin, with the sequence Asp-Pro inserted between the soluble protein and the toxic peptide.

The dp-pt or ps-dp-pt expression system of the present invention makes it possible to overproduce toxic proteins in host cells, in particular hydrophobic proteins, especially peptides which correspond to, or which comprise, hydrophobic domains of membrane-anchored proteins which may involve, for example, a membrane protein or a domain of a membrane protein. It may involve, for example, a protein of a virus, for example of a hepatitis C virus, of an AIDS virus, or of any other virus that is pathogenic for humans and, in general, for mammals.

For example, the dp-pt or ps-dp-pt system of the invention makes it possible to overproduce, in a host such as E. coli, the transmembrane domains of the E1 and E2 proteins of the hepatitis C virus, called TME1 and TME2, corresponding respectively to the sequences -- see Original Patent.

The nucleotide sequences that can be used for constituting the dp-pt system of the invention encoding the TME1 (dp-pt.sub.(TME1)) or TME2 (dp-pt.sub.(TME2)) proteins can be any of the possible sequences encoding respectively the DP-TME1 and DP-TME2 fusion proteins. The sequences encoding the TME1 and TME2 proteins may advantageously be, for example, SEQ ID NO: 3 and SEQ ID NO: 4, respectively, of the attached sequence listing. To obtain the dp-pt system, the dp sequence encoding the dipeptide Asp-Pro (DP) is added to these sequences.

The nucleotide sequences that can be used for constituting the ps-dp-pt system of the invention encoding the TME1 (ps-dp-pt.sub.(TME1)) or TME2 (ps-dp-pt.sub.(TME2)) proteins may be any of the possible sequences encoding the Ps-DP-TME1 and Ps-DP-TME2 fusion proteins, respectively. They may advantageously be, for example, the sequences ID No. 34, ID No. 35 and ID No. 36 of the attached sequence listing for TME1, making it possible to obtain a Ps-DP-TME1 chimeric protein. They may advantageously be, for example, the sequences ID No. 37, ID No. 38 and ID No. 39 of the attached sequence listing for TME2, making it possible to obtain a Ps-DP-TME2 chimeric protein.

In fact, the abovementioned nucleotide sequences have optimized codons for the expression of TME1 and TME2 in a bacterium, for example in E. coli.

A large number of HCV RNA sequences producing an infectious phenotype exist: these sequences can also be used in the present invention.

The sequence encoding the dipeptide Asp-Pro may be, for example: gacccg, or any other sequence encoding this dipeptide.

The sequence encoding GST may be, for example, that present in the pGEXKT plasmids, the sequence of which corresponds to SEQ ID NO: 29 of the attached sequence listing, or any equivalent sequence, i.e. encoding this soluble protein. The sequence encoding TrX may be, for example, that present in the pET32a+ expression plasmid, the sequence of which corresponds to SEQ ID NO: 30 of the attached sequence listing, or any equivalent sequence, i.e. encoding this soluble protein.

For the production of the toxic protein, the dp-pt or ps-dp-pt expression system of the invention is placed inside a host cell, for example by cloning in an appropriate plasmid, by means of the usual techniques for transforming a host in genetic recombination techniques.

The plasmid into which the expression system of the present invention may be cloned so as to form this vector will be chosen in particular according to the host cell. It may be, for example, the pT7-7 plasmid (SEQ TD NO: 33 of the attached sequence listing), a plasmid of the pGEX series (for example of SEQ ID NO: 31 of the attached sequence listing), sold for example by the company Pharmacia, or a plasmid of the pET32 series (for example of sequence ID No: 32 of the attached sequence listing), sold for example by the company Novagen.

The plasmids of the pGEX series and of the pET32 series will advantageously be used for implementing the present invention. In fact, they already comprise a ps sequence encoding a soluble protein (Ps), respectively glutathione S-transferase and thioredoxin. Thus, advantageously, the dp-pt system will be cloned into these plasmids downstream of this ps sequence encoding the soluble protein.

The present invention therefore also relates to an expression vector comprising a dp-pt or ps-dp-pt expression system according to the invention; in particular, a vector comprising a dp-pt expression system according to the invention and the oligonucleotide sequence of the pT7-7 plasmid, or a vector comprising a ps-dp-pt expression system according to the invention and the oligonucleotide sequence of a pGEX plasmid or of a pET32 plasmid.

For example, the expression vectors of the present invention that are suitable for a bacterial host such as E. coli and that allow overexpression of the abovementioned TME1 membrane protein may advantageously have an oligonucleotide sequence chosen from the sequences ID No. 40 (with pGEXKT), ID No. 42 (with pET32a+) and ID No. 44 (with PT7-7) of the attached sequence listing.

For example, the expression vectors of the present invention that are suitable for a bacterial host such as E. coli and that allow overexpression of the abovementioned TME2 membrane protein may advantageously have an oligonucleotide sequence chosen from the sequences ID No. 41 (with pGEXKT), ID No. 43 (with pET32a+) and ID No. 45 (with pT7-7) of the attached sequence listing.

In fact, the abovementioned expression vectors have codons that are optimized for the expression of the chimeric proteins of the present invention, including TME1 and TME2, in a bacterium, for example in E. coli.

The present invention also relates to a prokaryotic cell transformed with an expression vector according to the invention. This prokaryotic cell transformed with the expression vector of the present invention should preferably allow overexpression of the toxic protein for which the vector codes. Thus, any host cell capable of expressing the expression vector of the present invention can be used, for example E. coli, advantageously the E. coli strain BL21(DE3)pLysS.

The present invention also relates to a method for producing a toxic protein by genetic recombination, comprising the following steps: transforming a host cell with an expression vector according to the invention, culturing the transformed host cell under culture conditions such that it produces a fusion protein comprising the dipeptide Asp-Pro followed by the peptide sequence of the toxic protein from said expression vector, and isolating said fusion protein, and cleaving said fusion protein so as to recover the toxic protein.

The steps for transforming, culturing and isolating the chimeric protein produced can be carried out by means of the usual techniques of genetic recombination, for example by means of techniques such as those that are described in document [25].

The step consisting in isolating the fusion protein can be carried out by means of the usual techniques known to those skilled in the art for isolating a protein from a cell extract.

The fusion protein produced by means of the method of the invention has a "soluble protein-Asp-Pro-toxic protein" sequence. In the present description, the dipeptide Asp-Pro is also called DP according to the one-letter amino acid code.

For example, when the toxic protein is TME1, the fusion protein may have the SEQ ID NO: 46 of the attached sequence listing, which corresponds to the GST-DP-TME1 fusion protein; the SEQ ID NO: 48 of the attached sequence listing, which corresponds to the TrX-DP-TME1 fusion protein; or the SEQ ID NO: 50 of the attached sequence listing, which corresponds to the M-DP-TME1 fusion protein of the attached sequence listing.

For example, when the toxic protein is TME2, the fusion protein may have the SEQ ID NO: 47 of the attached sequence listing, which corresponds to the GST-DP-TME2 fusion protein; the SEQ ID NO: 49 of the attached sequence listing, which corresponds to the TrX-DP-TME2 fusion protein; or the SEQ ID NO: 51 of the attached sequence listing, which corresponds to the M-DP-TME2 fusion protein of the attached sequence listing.

The step consisting of cleavage of this fusion protein can advantageously be carried out by means of formic acid, which cleaves the fusion protein at the dipeptide Asp-Pro. It may be carried out, moreover, by means of any appropriate technique known to those skilled in the art for recovering a protein from a sample using a fusion protein.

The inventors are the first to have found a system that is really effective for producing and even overproducing, in particular in the Escherichia coli (E. coli) bacterium, hydrophobic peptides corresponding to the membrane domains of the E1 and E2 proteins of the hepatitis C virus envelope, the expression of which is lethal for the microorganism.

The field of application of the present invention concerns mainly the production of hydrophobic peptides on a large scale, in particular for fundamental and industrial research. Tn addition, the production of the chimeric protein consisting of the soluble protein, of the dipeptide Asp-Pro and of the hyrophohic peptide can be used for a functional purpose, in particular for obtaining information on the degree of oligomerization of the membrane domain or else on its heteropolymerization capacity.

The fusion proteins, or chimeric proteins, are produced via their coding DNA present, for example, in commercial plasmids and following which is introduced, in phase, the DNA encoding the Asp-Pro sequence followed by that encoding the toxic peptide. This application can be commercialized in the form of bacterial expression plasmids which will include the sequence of the Asp Pro site, downstream of that of the soluble proteins already present. The corresponding plasmid will be described, for example, as a tool that facilitates the production, via the biological pathway, of toxic membrane peptides or proteins.

Thus, the present invention is applicable to any system for overexpressing recombinant proteins, with or without fusion to a soluble protein such as, for example, GST or thioredoxin, including a non-natural Asp-Pro sequence inserted upstream of a sequence encoding a toxic domain of the protein, for example a membrane domain of a protein.
 

Claim 1 of 13 Claims

1. An expression system comprising a DNA sequence, wherein said DNA sequence encodes a fusion protein comprising a sequence selected from the group consisting of SEQ ID NOS: 46-51.

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