WO2017209280A1 - Novel protein identification method using yeast and fragmented cdna library - Google Patents

Abstract

The purpose of the present invention is to provide a novel method for identifying a protein in a yeast nucleus. More specifically, the present invention relates to a protein identification method which comprises: in a yeast nucleus, expressing a fusion protein comprising a nuclear localization signal, a DNA-binding protein and a bait protein, and another fusion protein comprising a nuclear localization signal, a transcriptional activation protein and a prey protein encoded by a fragmented partial cDNA, said prey protein lacking a signal peptide, a cell membrane or intracellular organelle localization sequence or a transmembrane domain; and then detecting the binding of the bait protein to the prey protein using the activity, luminescence, etc. of a reporter gene as an indication.

Description

A new method for protein identification using yeast and fragmented cDNA libraries.

The present invention relates to a protein identification method using, for example, yeast and a fragmented cDNA library.

It is known that the association or binding of a plurality of molecules inside and outside the cell acts as a biological signal that controls cell proliferation, differentiation, adhesion, and the like. In particular, identification of receptor molecules on cell membranes is useful for elucidating the physiological effects of various ligand molecules such as hormones. For these purposes, a method of concentrating a protein complex containing a receptor molecule by immunoprecipitation and performing mass spectrometry, a panning method using cultured cells, and the like are becoming common. However, many “orphan ligands” whose receptors are unknown are still known. Receptor identification has attracted attention because it is highly likely to be associated with various disease pathologies, but has not progressed due to the complexity of screening methods and lack of detection sensitivity.

On the other hand, the yeast two-hybrid method is known as a method for identifying binding of intracellular (cytoplasmic) molecules (Non-Patent Documents 1 to 3). The yeast two-hybrid method uses a gene vector to add a known gene to which a nuclear translocation signal has been added and a full-length cDNA chimeric gene to which the nuclear translocation signal has been added (usually reverse transcribed with an oligo dT primer in a commercial product) When the types are expressed and a bond between molecules is formed in the nucleus, the bond between the molecules is detected by the activity of the reporter gene whose expression is induced, light emission, or the like.

In the yeast two-hybrid method, when a gene encoding a membrane protein such as a ligand molecule or a receptor is inserted into a vector, the chimeric gene is nucleated by expression of the signal peptide at the 5 'end or the transmembrane region near the 3' end. The yeast two-hybrid method is not suitable for analysis of ligand-receptor binding.

In addition, many modifications of the yeast two-hybrid method have been reported. For example, the cells to be used can be changed to organisms other than yeast, or can be improved by developing a reporter gene portion. However, conventionally, a modification of the yeast two-hybrid method, which is intended to be detected in the nucleus as a partial region library of (membrane) protein and to detect the interaction that originally occurs outside the nucleus, is known. There wasn't.

As mentioned above, the conventional yeast two-hybrid method is not suitable for analysis of ligand-receptor binding due to the expression of the signal peptide at the 5 ′ end and the transmembrane region near the 3 ′ end. Therefore, the present invention aims to provide a method suitable for analyzing the binding between proteins containing a ligand-receptor by modifying the yeast two-hybrid method.

As a result of earnest research to solve the above problems, unlike the conventional yeast two-hybrid method (preparation by reverse transcription with oligo dT primer), random primers are used to create cDNA libraries from any location. Using a fragmented cDNA library in which reverse-transcribed cDNA or cDNA that has been intentionally fragmented by sonication or the like is inserted into a vector, the fragmented cDNA library can be obtained by performing the yeast two-hybrid method. Contains a clone lacking the signal peptide at the 5 'end and the transmembrane region near the 3' end even if it is a membrane protein, allowing stable expression in the nucleus, In this process, the binding between the ligand and the extracellular part of the receptor is reproduced in the nucleus, and in the process of detecting the binding using the reporter gene, binding analysis that could not be performed by the conventional method becomes possible. Which resulted in the completion of the Akira.

That is, the present invention includes the following.
(1) A protein identification method comprising a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein the first vector comprises a gene encoding a nuclear translocation signal and a DNA binding protein A gene encoding a decoy protein and a second vector comprising a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, The fragmented partial cDNA encodes a prey protein lacking a signal peptide, a cell membrane or organelle localization sequence or a transmembrane region, and in the nucleus of the yeast transformant, A fusion protein comprising a translocation signal, a DNA binding protein and a decoy protein, and said translocation signal and transcription-activating protein Fusion protein was expressed containing the prey proteins encoded by fragmented partial cDNA, the binding of the bait protein and 該獲 product protein, detecting the activity of the reporter gene as an index, said method.
(2) The method according to (1), wherein the decoy protein is a ligand and the prey protein is a receptor protein.
(3) The method according to (1), wherein the decoy protein is a protein encoded by a partial cDNA excluding various localization signal sequences.
(4) cDNA having a length of 100 to 1800 bp obtained by random prime reverse transcription.
(5) The cDNA according to (4), wherein the length is 100 to 1000 bp.
(6) A vector comprising a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, wherein the fragmented partial cDNA is described in (4) or (5) The vector, which is cDNA.
(7) A reagent kit for the protein identification method according to any one of (1) to (3), comprising the cDNA according to (4) or (5) or the vector according to (6).

This specification includes the disclosure of Japanese Patent Application No. 2016-112107, which is the basis of the priority of the present application.

According to the present invention, it is possible to contribute to the elucidation of signal molecules involved in various diseases by elucidating the receptors for orphan ligands and searching for and identifying molecules to which membrane proteins bind.

The protein identification method according to the present invention (hereinafter referred to as “the present method”) includes a step of culturing a yeast transformant into which the first and second vectors have been introduced, wherein
The first vector includes a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a bait protein,
The second vector includes a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA. The fragmented partial cDNA is a signal peptide, a cell membrane, or an organelle. Encodes a prey protein lacking a localization sequence or transmembrane region, and
In the nucleus of the yeast transformant, a fusion protein comprising the nuclear translocation signal, a DNA binding protein, and a decoy protein, and a prey encoded by the partial cDNA fragmented with the nuclear translocation signal, the transcriptional activation protein A fusion protein containing a protein is expressed, and the binding between the decoy protein and the prey protein is detected using the activity of a reporter gene as an indicator,
Is the method.

In general, in the conventional yeast two-hybrid method, a fusion protein of a DNA-binding protein and a molecule of interest (bait protein) and a cDNA library (derived from) a transcription library and a full-length cDNA (prey protein) When the fusion protein is expressed in the yeast nucleus and binding of the decoy protein to the library-derived molecule occurs, the activity of the reporter gene that induces expression is used as an index to bind the decoy protein to the library-derived molecule. Is detected.

The yeast two-hybrid method has become widespread as an inexpensive large-scale screening technique using the binding of two molecules as an index. However, when a protein derived from a cDNA library contains a signal peptide, a sequence that localizes to cell membranes or various organelles, the molecule cannot be expressed in the nucleus, and a bond with a decoy protein is formed in the nucleus. And reporter gene expression cannot be detected. For this reason, the yeast two-hybrid method is generally used as a technique for detecting the binding between intracellular proteins. In addition, a library commercially available in the prior art is one in which a cDNA that has been reverse-transcribed and synthesized from the 3 ′ end of mRNA using an oligoolidT primer (substantially full length) is inserted.

On the other hand, since this method uses a cDNA library with intentionally fragmented cDNA inserted, partial regions of molecules including membrane proteins and various localized sequences that were missed in the conventional method are expressed in the yeast nucleus. By doing so, binding to a decoy protein can be detected, and ligand-receptor binding that usually occurs outside the cell and association of membrane proteins can be detected inside the yeast nucleus, both inside and outside the cell.

In this method, first, first and second vectors are prepared.

The first vector includes a fusion gene in which a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a decoy protein are functionally linked. The first vector may be any vector that functions in yeast, and examples thereof include pBTM116, pGBKT7, and pGBT9.

In the first vector, in order to transfer the fusion protein encoded by the fusion gene comprising the gene encoding the DNA binding protein and the gene encoding the decoy protein into the nucleus of the yeast, a nuclear transfer signal is sent. Encoding gene is included in the fusion gene or added in advance (eg, Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1), LexA protein endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2), etc.) Must have been.

Examples of the DNA binding protein include a DNA binding domain (DBD) of Gal4 protein, a DBD of LexA protein, and the like.

In addition, examples of decoy proteins include orphan ligands whose receptors are unknown, proteins encoded by partial cDNAs excluding various localization signals (such as transmembrane domains and signal sequences localized to organelles) Is mentioned.

Thus, in the first vector, for example, in the direction from the N-terminal to the C-terminal, each gene is included in a functionally linked form in order to express a fusion protein including a nuclear translocation signal-DBD-bait protein. .

The second vector includes a fusion gene in which a gene encoding a nuclear translocation signal, a gene encoding a transcription activation protein, and a fragmented partial cDNA are functionally linked. The second vector may be any vector that functions in yeast, and examples thereof include pACT2, pGADT7, and pGAD10.

In the second vector, in order to transfer the fusion protein encoded by the fusion gene comprising the gene encoding the transcriptional activation protein and the fragmented partial cDNA into the yeast nucleus, The transfer signal must be endogenous or previously added (for example, Large T antigen residuesres47 to 54 (PKKKRKVE: SEQ ID NO: 1), LexA protein endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2), etc.) Don't be.

In addition, the transcription activation protein includes a DNA binding protein encoded by the fusion gene contained in the first vector and transcription activity when located close to a reporter gene control sequence (promoter etc.) in yeast. And activates transcription of a reporter gene, such as an activator domain (AD) of Gal4 protein corresponding to DBD of Gal4 protein.

On the other hand, the fragmented partial cDNA is different from a full-length gene cDNA library prepared using an oligo dT primer, for example, a known cDNA library preparation (e.g., cDNA Library Construction Kit (Takara Bio Inc.)). In preparation of a cDNA library using a kit), using an mRNA collected from an arbitrary organism or tissue and a random primer (for example, a random primer comprising the base sequence described in SEQ ID NO: 3 used in the following Examples) A cDNA library reversely transcribed from an arbitrary location, or a cDNA library intentionally fragmented by cutting with ultrasonic treatment or the like. The cDNA obtained by random prime reverse transcription has, for example, a length of 100 to 1800 bp (preferably 100 to 1000 bp). A cDNA library fragmented by cleavage using sonication or the like is prepared, for example, by adding an EcoRI adapter to the end and binding it to a large excess of vector having an EcoRI stump. Such partial cDNAs include those encoding a prey protein that binds to a ligand or membrane protein, lacking a signal peptide, a cell membrane or organelle localization sequence or a transmembrane region. Examples of the prey protein include a receptor protein.

Thus, in the second vector, for example, in the direction from the N-terminal to the C-terminal, a fusion protein containing a nuclear protein, a prey protein encoded by a Gal4 activator domain (AD) -fragmented cDNA is sequentially expressed. Each gene in a functionally linked form.

In this method, the first and second vectors are then transformed into yeast to produce yeast transformants that express the first and second vectors. Examples of yeast include budding yeast (Saccharomyces cerevisiae). In the yeast nucleus, transcription is activated by the DNA binding protein encoded by the fusion gene contained in the first vector and the transcription activation protein encoded by the fusion gene contained in the second vector. There are control sequences to be converted and a reporter gene located downstream thereof. Examples of the control sequence include a base sequence called UASG (UpstreamUpActivation Sequences for galactose) for the Gal4 protein. Examples of the reporter gene include a gene encoding β-galactosidase and HIS3. The control sequence and the reporter gene located downstream thereof are present in the yeast nucleus because they are integrated into the genomic DNA.

The method for introducing the first and second vectors into yeast is not particularly limited as long as it is a method for introducing DNA into yeast, and examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.

Next, the obtained yeast transformant is cultured under conditions that allow it to grow. In the nucleus of the yeast transformant, a fusion protein comprising a nuclear translocation signal, a DNA-binding protein and a decoy protein, and a prey protein encoded by the nuclear translocation signal, a transcriptional activation protein and a fragmented partial cDNA The decoy protein and the prey protein interact with each other so that the DNA-binding protein and the transcriptional activation protein are close to each other and bind to a control sequence located upstream of the reporter gene. , Transcription of the reporter gene is promoted. Therefore, the binding of the decoy protein and the prey protein can be confirmed using the activity of the reporter gene as an index, and the prey protein corresponding to the decoy protein can be identified.

In culturing yeast transformants, the temperature is set to, for example, 25 to 30 ° C., and the pH of the medium is, for example, pH 5 so that the transformants grow and the reporter protein encoded by the reporter gene is not inactivated. Set at around .8 and continue culturing until the required amount of cells for each assay is obtained.

In a yeast transformant in which the activity of the reporter gene is confirmed, the gene fragment encoding the partial prey protein can be identified by determining the sequence of the fragmented partial cDNA contained in the second vector. . Furthermore, by comparing the gene fragment encoding the identified partial prey protein with, for example, a gene sequence registered in a known database, the gene encoding the prey protein (if registered) or the gene concerned The full-length prey protein encoded by can also be identified.

According to this method, the detection sensitivity of binding (up to about 1 μM Kd binding is expected to be detected by using the yeast reporter gene system, compared to the case of panning in cultured cells. Improved S / N ratio, faster screening (normally about 14 days are required to determine the binding of about 1 million clones), stability of recovered genes, faster cloning, economy, etc. Be expected. In addition, receptor identification of orphan ligands may be directly linked to drug discovery, and this method can be applied to receptor identification of orphan ligands. Furthermore, the present method can be widely applied to the binding screening between membrane proteins.

The present invention also relates to a reagent kit for the present method comprising the cDNA itself obtained by the above-mentioned random prime reverse transcription, the second vector itself, the cDNA obtained by the random prime reverse transcription or the second vector. The reagent kit can further include, for example, a buffer and a container used in the present method, instructions for use, and the like.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to these Examples.
1. Synthesis of Fragmented cDNA Fragmented cDNA was synthesized using the method of cDNA Library Construction Kit (Takara Bio Inc.) with partial modification.

(1) Synthesis of 1st strand cDNA Primer with 4 kinds of 4 nucleotides of dATP, dCTP, dGTP and dTTP randomly added to the 3 'side of the restriction enzyme XhoI recognition sequence (5'-CTCGAG-3') ( Sequence 5′-TAGAACTCGAGNNNNNN-3 ′ (SEQ ID NO: 3); hereinafter referred to as “random primer” (600 μM, 1 μl), 5 μg of mRNA collected from any organism or tissue, and dNTP mixture (dATP , 5-methyl dCTP, dGTP, dTTP) (10 mM each) was mixed in a microcentrifuge tube and made up to 10 μl with RNase-free water. This was heated at 65 ° C. for 5 minutes and denatured by rapid cooling on ice. To this, reverse transcriptase (PrimeScript RTase Takara Bio Inc.) (200 U / μl) 1 μl, RNase inhibitor (40 U / μl) 1 μl, reaction buffer (250 mM Tris-HCl (pH 8.3 ), 375 mM KCl, 15 mM MgCl ₂ ) 4 μl and RNase-free water 4 μl were added to make 20 μl, and a reverse transcription reaction was performed at 42 ° C. for 1 hour. After 1 hour, it was cooled on ice for 2 minutes. The cDNA synthesized by this reaction was designated as 1st strand cDNA.

(2) 2nd strand cDNA synthesis 1st strand cDNA is mixed with dNTP mixture (dATP, dCTP, dGTP, dTTP) 4.5 μl, RNaseH and DNA ligase mixture 2 μl, DNA polymerase I (20 U / μl) 2 μl, 30 μl of reaction buffer and 87.5 μl of RNase-free water were added to make 146 μl, followed by reaction at 16 ° C. for 2 hours. After 2 hours, the mixture was allowed to stand at 70 ° C. for 10 minutes and then allowed to stand at room temperature for 5 minutes or more. The double-stranded cDNA synthesized by this reaction was designated as 2nd strand cDNA.

(3) Smoothing of the ends of the 2nd strand cDNA 4 μl of T4 DNA polymerase (1 U / μl) was added to the 2nd strand cDNA and reacted at 37 ° C. for 10 minutes to smooth the ends of the 2nd strand cDNA. An equal amount of phenol / chloroform / isoamyl alcohol (25: 24: 1 mixed solution) was added thereto, mixed well, centrifuged, and the upper layer was transferred to a new microcentrifuge tube. An equal amount of chloroform / isoamyl alcohol (24: 1 mixed solution) was added thereto, and after mixing well, the mixture was centrifuged and the upper layer was transferred to a new microcentrifuge tube. 1/10 volume of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of ethanol were added to this, and after mixing well, the mixture was centrifuged at 15,000 rpm for 30 minutes at room temperature, and the supernatant was removed. After adding 200 μl of 70% ethanol to the precipitate and rinsing the precipitate, the mixture was centrifuged at 15,000 rpm for 3 minutes at room temperature, and the supernatant was removed. After drying the precipitate, 12.5 μl of RNase-free water was added to dissolve the precipitate. This was used as the end-blunted 2nd strand cDNA.

(4) Addition of EcoRI adapter to end-blunted 2nd strand cDNA Add 12.5 μl of end-blunted 2nd strand cDNA solution to EcoRI-SmaI adapter (5′-AATTCCCGGG-3 ′ single strand with 5 ′ end dephosphorylated) DNA (SEQ ID NO: 4) and 5′-CCCGGG-3 ′ single-stranded DNA annealed) (0.4 μg / μl) 3.5 μl and T4 DNA ligase (350 U / μl) 2 μl 2 μl of working buffer was added to make a total volume of 20 μl. This was mixed well and then reacted at 8 ° C. overnight or longer. After the reaction, the mixture was allowed to stand at 70 ° C. for 30 minutes to inactivate T4 DNA ligase, and then allowed to stand at room temperature for another 5 minutes. This was designated as adapter-added 2nd strand cDNA.

(5) Cleavage of adapter-added 2nd strand cDNA with restriction enzyme XhoI 20 μl of adapter-added 2nd strand cDNA solution, 3 μl of XhoI (10 U / μl) and reaction buffer (500 mM Tris-HCl (pH 7.5)) ), 100 mM MgCl ₂ , 10 mM dithiothreitol, 1 M NaCl) 5 μl, 0.1 μl BSA (bovine serum albumin) 5 μl, and RNase-free water 17 μl were added to make a total volume of 50 μl. This was mixed well and then reacted at 37 ° C. for 3 hours. In this reaction, the XhoI recognition sequence in the random primer is cleaved, but the XhoI recognition sequence in the cDNA is not cleaved by XhoI because 5-methyl dCTP is used during the synthesis of the first strand cDNA.

(6) Removal of short-chain DNA Using gel filtration or silica-based beads, unreacted adapters and short-chain DNAs of several tens of residues or less are removed. 2nd strand cDNA cleaved with XhoI is applied to a gel filtration column (select the gel type according to the size of the target cDNA) equilibrated with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). The mixture was added and centrifuged (conditions depend on the column). By this centrifugation operation, cDNAs of a certain size or larger were obtained, which did not contain unreacted EcoRI-SmaI adapters or short DNA fragments cleaved with XhoI. An equal amount of phenol / chloroform / isoamyl alcohol (25: 24: 1 mixed solution) was added thereto, mixed well, centrifuged, and the upper layer was transferred to a new microcentrifuge tube. An equal amount of chloroform / isoamyl alcohol (24: 1 mixed solution) was added thereto, and after mixing well, the mixture was centrifuged and the upper layer was transferred to a new microcentrifuge tube. 1/10 volume of 3 M sodium acetate buffer (pH 5.2) and 2.5 volumes of ethanol were added to this, and after mixing well, the mixture was centrifuged at 15,000 rpm for 30 minutes at room temperature, and the supernatant was removed. After adding 200 μl of 70% ethanol to the precipitate and rinsing the precipitate, the mixture was centrifuged at 15,000 rpm for 3 minutes at room temperature, and the supernatant was removed. After drying the precipitate, 15 μl of RNase-free water was added to dissolve the precipitate, which was designated as EcoRI-XhoI-cleaved cDNA.

(7) Binding of EcoRI-XhoI-cleaved cDNA to a vector A vector according to purpose was cleaved with EcoRI and XhoI, and EcoRI-XhoI-cleaved cDNA was ligated using DNA ligase.

Next, the vector and EcoRI-XhoI-cleaved cDNA combined were introduced into E. coli by electroporation, and any 10 clones were selected and analyzed for the size of the fragmented cDNA inserted by PCR. After confirming that it was 100 to 1000 bases, a cDNA library was recovered from E. coli.

2. Screening of mouse brain-derived molecules Saccharomyces cerevisiae transformed with pBTM116-HA-KM-hENHO, a library in which cDNA prepared based on mRNA recovered from mouse brain was inserted into vector pACT2 according to Section 1 above. It was introduced into L40 and seeded on SD-Leu-Trp-His + 3-AT medium (3-AT = 3-amino-1,2,4-triazole). Here, the vector pACT2 into which the cDNA is inserted has a gene encoding the Large T antigen residues 47 to 54 (PKKKRKVE: SEQ ID NO: 1) in order from the 5 ′ end to the 3 ′ end and an activator domain (AD) of the Gal4 protein. And a prepared cDNA. In addition, the vector pBTM116-HA-KM-hENHO contains an endogenous nuclear localization signal sequence (KRLKK: SEQ ID NO: 2) and DNA binding domain (DBD) of the LexA protein in order from the 5 ′ end to the 3 ′ end. It has a gene that encodes it and a gene that encodes human adropin (hENHO) as a gene that encodes a decoy protein. Furthermore, Saccharomyces cerevisiae L40 has a gene (reporter gene) encoding β-galactosidase and an HIS3 gene under the control of UASG downstream. When molecules expressed from the two plasmids are combined in the cell, yeast cells can grow even in a His-deficient medium due to the expression of the HIS3 gene.

The seeded medium was transferred to a 30 ° C. incubator and cultured for several days until colonies were visible. Colonies were transferred to fresh SD-Leu-Trp medium and cultured in a 30 ° C. incubator. Using a part of the grown yeast, β-galactosidase assay was performed, and a group of clones positive for β-galactosidase activity was analyzed. As a result, those containing partial cDNA of membrane protein were included.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims (7)

1. A method for identifying a protein comprising the step of culturing a yeast transformant into which the first and second vectors have been introduced,
   The first vector includes a gene encoding a nuclear translocation signal, a gene encoding a DNA binding protein, and a gene encoding a decoy protein,
   The second vector includes a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA. The fragmented partial cDNA is a signal peptide, a cell membrane, or an organelle. Encodes a prey protein lacking a localization sequence or transmembrane region, and
   Prey encoded in the nucleus of the yeast transformant by a fusion protein comprising the nuclear translocation signal, a DNA-binding protein and a decoy protein, and a partial cDNA fragmented with the nuclear translocation signal, a transcriptional activation protein A fusion protein containing a protein is expressed, and the binding between the decoy protein and the prey protein is detected using the activity of a reporter gene as an indicator,
   Said method.
2. The method according to claim 1, wherein the decoy protein is a ligand and the prey protein is a receptor protein.
3. The method according to claim 1, wherein the decoy protein is a protein encoded by a partial cDNA excluding various localization signal sequences.
4. A cDNA having a length of 100 to 1800 bp obtained by random prime reverse transcription.
5. The cDNA according to claim 4, wherein the length is 100 to 1000 bp.
6. A vector comprising a gene encoding a nuclear translocation signal, a gene encoding a transcriptional activation protein, and a fragmented partial cDNA, wherein the fragmented partial cDNA is the cDNA according to claim 4 or 5. vector.
7. A reagent kit for the protein identification method according to any one of claims 1 to 3, comprising the cDNA according to claim 4 or 5 or the vector according to claim 6.