JP7453745B2 - Method for screening peptides that inhibit the activity of toxic proteins and methods for producing toxic proteins - Google Patents

Description

The present invention relates to a method for screening for peptides that inhibit the activity of toxic proteins and a method for producing toxic proteins.

Conventionally, protein expression systems have been known in which a gene encoding a target protein is introduced into host cells such as bacteria, yeast, insect cells, mammalian cells, etc. to express the target protein, and these systems are used for structural and functional analysis of proteins. has been done. However, when such an expression system is used to express a toxic protein, there are cases in which transformants cannot be obtained in sufficient quantities or at all due to toxicity to host cells, making it difficult to perform protein synthesis.

Several methods have been proposed so far for expressing toxic proteins using protein expression systems. First, there is a known method to suppress the basal expression of a toxic protein before inducing its expression, thereby reducing its toxicity. A method for controlling and suppressing the basal expression of toxic proteins in E. coli before induction of expression (Non-patent Document 1); A method for suppressing the basal expression of a toxic protein before inducing its expression (Non-Patent Document 2) has been reported. Furthermore, a method using an E. coli strain that is resistant to toxicity has been reported (Non-Patent Document 3). However, these methods cannot be applied to proteins with strong toxicity. Additionally, a method using a cell-free synthesis system (Patent Document 1) has been reported, but because the synthesis uses research reagents, the cost per yield is high and it is not suitable for mass production. Furthermore, by changing the biological species of the host cell, it may be possible to avoid the toxicity that occurred in the original host cell, but selecting an appropriate host cell is time-consuming and complicated. Therefore, from the viewpoint of industrial production, there is a need for the development of a technique for easily carrying out mass production using host cells such as E. coli that can be cultured in large quantities.

On the other hand, in the development of protein inhibitors, a method is known in which a vast number of compound libraries are screened for compounds that exhibit inhibitory ability against target proteins. However, it requires isolation and purification of the target protein, a large amount of money for library construction and maintenance, and a lot of manpower for screening. Additionally, phage display methods and the like (Non-Patent Document 4) are known to search for peptides that bind to target proteins, but although molecules that bind to target proteins can be obtained, it is not guaranteed that they will have inhibitory ability. It is necessary to separately evaluate the inhibitory ability. Therefore, a method that can simply and efficiently search for protein inhibitors is desired.

Pierisin-1 is a protein with a total length of about 98 kDa that was discovered in the pupa of the cabbage butterfly. Consists of an N-terminal domain that is a catalytic domain, an autoinhibitory peptide, and a C-terminal domain that is responsible for binding to glycolipids on the cell membrane. Using NAD ⁺ as a coenzyme, it ADP-ribosylates guanosine in DNA and leads cells to apoptosis. It is an enzyme (Non-patent Documents 5, 6). What is noteworthy is its high cytotoxic activity. Piericin-1 purified from cabbage butterfly pupa exhibits extremely strong cytotoxic activity against various cultured cells, with IC ₅₀ values on the fM order, although the activity varies depending on the cell type (Non-Patent Document 5) . Therefore, pierisin-1 is a very attractive material as a seed for anticancer drugs.

International Publication No. 2016/068294

Mesa-Pereira B. et al. Sci Rep. 2017, 7(1): 3069 Studier F.W. J. Mol. Biol. 1991, 219(1): 37-44 Dumon-Seignovert L. et al. Protein Expr Purif. 2004, 37(1): 203-206 Yuji Ito et al., Biophysics 2008, 48(5): 294-298 Kono T et al. Cancer Lett. 1999; 137(1): 75-81 Watanabe M et al. Proc. Natl. Acad. Sci. USA 1999; 96(19): 10608-10613

In order to express toxic proteins in host cells, it was thought that it was necessary to express them in a state where the activity of the toxic proteins was inhibited. Therefore, an object of the present invention is to screen for substances that inhibit the activity of toxic proteins, and to provide a method for producing toxic proteins using the substances.

Therefore, in view of the above-mentioned problems, the present inventors focused on the toxic protein Piericin-1 and peptides as substances that can inhibit the activity of Piericin-1, searched for inhibitory peptides, and utilized the inhibitory peptides. We conducted extensive studies to express piericin-1 in host cells. As a result, when host cells were transformed with a vector library containing vectors containing piericin-1, a protease recognition sequence, and a gene encoding an arbitrary peptide, and the transformed host cells were cultured, colonies were found. We have discovered that piericin-1 is obtained, that piericin-1 is expressed in the colony, and that the peptide in the vector introduced into the cells forming the colony functions as a peptide that inhibits the toxicity of piericin-1. Completed the invention.

That is, the present invention provides the following [1] to [11].
[1] Transforming a host cell with a vector library containing a gene encoding a toxic protein and any peptide, or a vector library containing a vector into which a gene encoding a toxic protein, a protease recognition sequence, and any peptide is integrated;
culturing the transformed host cells to obtain colonies;
A method of screening for a peptide that inhibits the activity of a toxic protein, the method comprising:
[2] The screening method of [1], wherein the host cell is E. coli.
[3] The screening method of [1] or [2], wherein the peptide has a length of 3 to 50 amino acids.
[4] The screening method of [1] to [3], wherein the peptide contains at least two cysteines.
[5] Transforming a host cell with a vector library containing a gene encoding a toxic protein and any peptide, or a vector library containing a vector into which a gene encoding a toxic protein, a protease recognition sequence, and any peptide is integrated;
culturing the transformed host cells to obtain colonies;
isolating the toxic protein;
A method for producing a toxic protein, comprising:
[6] The method for producing [5], wherein the host cell is E. coli.
[7] The method for producing [5] or [6], wherein the peptide has a length of 3 to 50 amino acids.
[8] The production method of [5] to [7], wherein the peptide contains at least two cysteines.
[9] Pierisin-1 produced by the production method of [5] to [8].
[10] Piericin containing as an active ingredient one or more selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, and a peptide consisting of the amino acid sequence of SEQ ID NO: 3. 1 toxicity inhibitor.
[11] A vector library containing a gene encoding a toxic protein and any peptide, or a vector into which a gene encoding a toxic protein, a protease recognition sequence, and any peptide has been integrated.

According to the method of the present invention, it is possible to easily and efficiently screen in host cells for peptides that inhibit the activity of toxic proteins that are difficult to express in conventional protein expression systems using host cells due to their toxicity. , the toxic protein can be expressed and produced in large quantities. The method of the present invention simultaneously expresses proteins and obtains inhibitors for toxic proteins for which it is difficult to perform inhibitor screening and drug discovery research is difficult due to the difficulty of expressing toxic proteins. It is very useful as a system that can do this.

FIG. 3 is a diagram showing protein expression by western blotting using the obtained colonies. The control was the N-terminal domain (residues 1-233) of piericin-1 synthesized using a cell-free synthesis system. It is a figure of the sequence analysis result using a forward primer. The part surrounded by a solid line is the piericin-1 sequence, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence. It is a figure of the sequence analysis result using a reverse primer. The part surrounded by a solid line is the piericin-1 sequence, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence. FIG. 3 is a diagram showing protein expression by western blotting using the obtained colonies. The control was the N-terminal domain (residues 1-233) of piericin-1 synthesized using a cell-free synthesis system. It is a figure of the sequence analysis result using a reverse primer. The part surrounded by a solid line is the piericin-1 sequence, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence. It is a figure of the sequence analysis result using a reverse primer. The part surrounded by a solid line is the piericin-1 sequence, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence.

In this specification, the identity (homology) of amino acid sequences and base sequences is determined by the Lipman-Pearson method (Lipman, D. J. and Pearson, W. R. 1985. Rapid and sensitive protein) using genetic information processing software GENETYX (manufactured by Genetics). A homology analysis (Search homology) program can be used. The parameters are Unit Size to compare=2, Pick up Location=5, and the results are displayed as a percentage.

Furthermore, the term "gene" is intended to include not only double-stranded DNA but also single-stranded DNA such as the sense strand and antisense strand that constitute it, and is not limited in any way to its length. Examples of polynucleotides include RNA and DNA, and DNA includes cDNA, genomic DNA, and synthetic DNA.

The method of the present invention has aspects of being a screening method for peptides that inhibit the activity of toxic proteins and aspects of being a method for producing toxic proteins, and includes genes encoding toxic proteins and arbitrary peptides, or toxic proteins. , a step of transforming a host cell with a vector library containing a vector into which a gene encoding a protease recognition sequence and an arbitrary peptide has been integrated (first step), and culturing the transformed host cell. and a step of obtaining colonies (second step). The method for producing a toxic protein further includes a step of isolating the toxic protein (third step).

In the method of the invention, a toxic protein and any peptide are introduced into a host cell and expressed simultaneously. When the arbitrary peptide has the function of inhibiting the activity of the toxic protein, the toxic protein with its activity inhibited is expressed in the host cell, and a colony of transformants can be obtained. On the other hand, if the arbitrary peptide does not have such a function, the toxic protein is expressed in the host cell while retaining its activity, resulting in the host cell dying and no colony being obtained. That is, in the method of the present invention, it is possible to obtain a peptide that exhibits inhibitory activity against a toxic protein based on the determination of whether the host cell is alive or dead, and it is also possible to express the toxic protein in the host cell. Here, "an arbitrary peptide inhibits the activity of a toxic protein" means that the arbitrary peptide interacts with the toxic protein and reduces or eliminates the activity of the toxic protein.

The first step of the method of the invention is to create a construct containing a gene encoding a toxic protein and an optional peptide, or a fusion protein consisting of two or three components: a toxic protein, a protease recognition sequence, and an optional peptide. , the construct is inserted into a vector for protein expression, and a host cell is transformed using the obtained vector library containing a plurality of vectors having different sequences of arbitrary peptides.

In the present invention, a toxic protein refers to a toxic protein that, when expressed in a host cell, causes some form of pathological change in the host cell, not only directly killing the cell but also causing DNA cleavage. Proteins that cause structural and functional damage to cells, such as the formation of base dimers, chromosome breakage, damage to the cell division apparatus, and reduction in various enzyme activities. Examples of toxic proteins include, but are not particularly limited to, degrading enzymes such as proteases and nucleases that are essential components for maintaining the life activities of host cells, bacteriocins such as colicin, and proteinaceous toxins such as diphtheria toxin. Also included are partial proteins such as the catalytic domain of toxic proteins, as long as they retain toxicity. In the method of the present invention, from the viewpoint of screening for peptides that inhibit the activity of toxic proteins, a toxic protein having an autoinhibitory peptide that inhibits the activity of the toxic protein is preferable, and in this case, a toxic protein that inhibits the activity more strongly than the autoinhibitory peptide. It is also possible to screen for peptides that
In the Examples described later, the toxic protein is Piericin-1 isolated from the pupa of the cabbage butterfly, specifically the N-terminal catalyst of Piericin-1 consisting of the amino acid sequence shown at positions 1 to 233 of the amino acid sequence of SEQ ID NO: 4. domain, but the toxic protein is not limited to this. Piericin-1 is an enzyme that ADP-ribosylates guanosine in DNA and leads cells to apoptosis. Piericin-1 is known to have high cytotoxic activity, and even if attempts were made to synthesize it by transforming E. coli using conventional genetic recombination techniques, it would be impossible to obtain transformants because the E. coli would die. , protein synthesis could not be performed. According to the method of the present invention, it is possible to screen for peptides that inhibit the activity of Piericin-1, and it is also possible to produce Piericin-1.

In the present invention, a protease recognition sequence refers to a protease recognition sequence that cleaves proteins in a sequence-specific manner. The protease is preferably a protease that exhibits activity in a temperature range (eg, 4 to 37°C) and a pH range (eg, pH 5.5 to 9.0) in which the protein structure is stable. Commercially available enzymes may be used as such proteases; for example, TEV protease ( Applied Biological Materials Inc.), HRV 3C protease (Takara Bio Inc.) which recognizes the sequence LEVLFQGP (SEQ ID NO: 8) consisting of 8 amino acids and cleaves between Q and G, and the like.

In the present invention, an arbitrary peptide is a peptide that has an arbitrary amino acid sequence and is a candidate for a peptide that inhibits the activity of a toxic protein. The length of the peptide is not particularly limited, but is usually 3 to 50 amino acids, preferably 5 to 40 amino acids, more preferably 8 to 30 amino acids, and particularly preferably 10 to 20 amino acids. The amino acid sequence constituting the peptide is not particularly limited, and assuming that the length of the peptide is 15 amino acids, theoretically any ²⁰¹⁵ amino acid sequences are possible. Among such peptides, peptides containing two or more cysteines (Cys) are preferable from the viewpoint of changing the folding of the peptide by taking advantage of the difference in the reducing environment, and the interval between Cys is 3 to 8 amino acids. More preferred are peptides in which the 1st, 7th, 13th and 17th amino acids are Cys.

A construct containing a gene encoding a toxic protein and any peptide, or a fusion protein consisting of two or three components: a toxic protein, a protease recognition sequence, and any peptide, may be produced by conventional genetic engineering techniques. For example, information on a base sequence encoding a toxic protein and a base sequence encoding any peptide, or a base sequence encoding a toxic protein, a base sequence encoding a protease recognition sequence, and a base sequence encoding any peptide. Forward primers and reverse primers can be easily obtained by designing a forward primer and a reverse primer and using the DNA of an organism that produces the toxic protein as a template by a conventional PCR method.
Here, a base sequence encoding a toxic protein and a base sequence encoding any peptide, or a base sequence encoding a toxic protein, a base sequence encoding a protease recognition sequence, and a base sequence encoding any peptide are expressed as Requires that it be possible to connect. "Expressibly linked" means that when a construct containing these base sequences is inserted into a vector containing an appropriate promoter and the vector is introduced into an appropriate host cell, toxic proteins and any Refers to the production of a fusion protein that includes a peptide, or a fusion protein that includes a toxic protein, a protease recognition sequence, and any peptide. Moreover, "linking" is a concept that includes both the case where the two or three base sequences described above are directly bonded, and the case where they are bonded via another base sequence. The order in which each base sequence is linked is as long as it includes a base sequence that encodes a protease recognition sequence, so long as the sequence is sandwiched between a base sequence that encodes a toxic protein and a base sequence that encodes any peptide. , not particularly limited. When constructing a construct, forward primers and reverse primers may be appropriately designed depending on the order of ligation.
An example of a more specific method for producing the construct will be described below, but the method for producing the construct is not limited thereto.

The base sequence of a gene encoding a toxic protein may be obtained by referring to DNA sequence information of an organism that produces the toxic protein, base sequence information from a known database, or the like. Genes encoding toxic proteins include, in addition to genes consisting of the base sequences obtained above, genes consisting of base sequences in which one or several bases have been deleted, inserted, substituted, or added, and which are toxic. Genes that encode proteins that have a base sequence that has an identity of 85% or more, preferably 90% or more, more preferably 95% or more with the base sequence, and that encode toxic proteins are also included. . Here, one or several pieces preferably means 1 to 20 pieces, more preferably 1 to 10 pieces.

The base sequence encoding the protease recognition sequence may be obtained by converting the amino acid sequence constituting the recognition sequence of the protease used into a base sequence. When converting an amino acid sequence into a base sequence, it is preferable to take into consideration the frequency of codon usage in the host cell used for transformation.

The base sequence encoding any peptide is any base sequence consisting of the number of bases corresponding to the length of the peptide, and the types of bases are not limited. When any peptide contains Cys, the base sequence at the corresponding position may be a codon sequence encoding Cys.

As the forward primer, an oligonucleotide consisting of a base sequence containing the start codon at the 5' end of the gene encoding the toxic protein can be suitably used. On the other hand, the reverse primer is a sequence complementary to a base sequence encoding any peptide, a sequence complementary to a base sequence excluding the stop codon at the 3' end of a gene encoding a toxic protein, or a sequence complementary to a base sequence encoding an arbitrary peptide. An oligonucleotide consisting of a sequence complementary to a base sequence, a sequence complementary to a base sequence encoding a protease recognition sequence, and a sequence complementary to a base sequence excluding the stop codon at the 3' end of a gene encoding a toxic protein. It can be suitably used. As a base sequence complementary to a base sequence encoding an arbitrary peptide in the reverse primer, each of the base sequences constituting the base sequence is selected so that the DNA fragment amplified by PCR from the base sequence is a mixture of possible base sequences. Preferably, the base is N, which is a mixture of adenine (A), thymine (T), guanine (G) and cytosine (C). Alternatively, as a base sequence complementary to a base sequence encoding an arbitrary peptide, a base sequence MNN, which is a complementary chain of codon NNK, may be repeated in order to reduce the probability of occurrence of a stop codon. Furthermore, in order to facilitate the incorporation of the construct into a vector, a sequence for restriction enzyme cleavage can be added to the 5' end of the primer. These primers may be chemically synthesized. The length of the primer is not particularly limited, but the length that anneals to the template is preferably 10 to 50 consecutive bases, more preferably 15 to 35 consecutive bases.

A construct containing a gene encoding a toxic protein and any peptide, or a fusion protein consisting of 2 or 3 components: a toxic protein, a protease recognition sequence, and any peptide, includes the above-mentioned forward primer and reverse primer as a set, It can be obtained by performing a PCR reaction using DNA of an organism that produces toxic proteins as a template. The construct obtained by PCR is thus a mixture of various constructs that differ in the sequence of any given peptide. Note that the PCR conditions may be determined as appropriate, taking into account the length of the primer, the length of the amplified fragment, the type of polymerase used, etc.

In the present invention, the vector is not particularly limited as long as it is replicable in a host cell, and examples thereof include a plasmid vector and a virus vector, but from the viewpoint of operability, a plasmid vector is preferred. The vector DNA used to construct the vector is preferably one that is widely used and easily available, and examples thereof include pUC19 (manufactured by Takara Bio Inc.), pTV118N (manufactured by Takara Bio Inc.), pGEX (manufactured by GE Healthcare Japan, Ltd.), pET160 (manufactured by Invitrogen), pDEST (manufactured by Invitrogen), and pET-coco-2 (manufactured by Merck Millipore).

The vector may include a DNA region that includes a DNA replication initiation region or origin of replication. Alternatively, in the above vector, a promoter region, terminator region, or signal region is placed upstream of the gene encoding the toxic protein and any peptide of the present invention, or the gene encoding the toxic protein, protease recognition sequence, and any peptide. may be operably linked. Here, the expression "operably linked" between a gene and a control sequence means that the gene and the control region are arranged in such a way that the gene can be expressed under the control of the control region. .
The types of control sequences such as the promoter region, terminator region, signal region, etc. are not particularly limited, and commonly used control sequences can be appropriately selected and used depending on the host to be introduced.
Alternatively, in the above vector, a gene encoding the toxic protein and any peptide of the present invention, or a gene encoding the toxic protein, protease recognition sequence, and any peptide may be added to facilitate isolation of the toxic protein. The tag sequences may be operably linked. Such tag sequences include, but are not particularly limited to, His tags, GST tags, GFP tags, HA tags, and the like.
Alternatively, the vector may further incorporate a marker gene (for example, a drug resistance gene for ampicillin, neomycin, kanamycin, chloramphenicol, etc.) for selecting a host into which the vector has been appropriately introduced. good. Alternatively, when an auxotrophic strain is used as the host, a gene encoding a required nutrient synthesizing enzyme may be incorporated into the vector as a marker gene. Alternatively, when using a selective medium that requires a specific metabolism for growth, a gene related to the metabolism may be incorporated into the vector as a marker gene.

Introduction of a gene encoding a toxic protein and any peptide, or a construct containing a gene encoding a toxic protein, protease recognition sequence, and any peptide into such a vector can be performed by conventional DNA recombination techniques. For example, vectors and constructs may be cut with appropriate restriction enzymes and ligated. Vectors containing the construct thus obtained can express genes encoding toxic proteins and arbitrary peptides, or genes encoding toxic proteins, protease recognition sequences, and arbitrary peptides, and vectors in which the sequences of arbitrary peptides are different from each other. It is a mixture of vector libraries (random vector libraries).

In the present invention, the host cell may be any cell capable of expressing a foreign gene. Examples of host cells include bacteria such as Escherichia coli and Bacillus subtilis, yeast, insect cells, mammalian cells, etc. Among these, Escherichia coli is preferred from the viewpoint of low cost, rapid proliferation, and easy control of expression. In addition, from the viewpoint of making it easier to determine whether the host cell is alive or dead after transformation, when a gene encoding a toxic protein is introduced into the host cell, the life activity of the host cell is partially or completely inhibited. Cells are preferred.

To transform host cells using a vector library, methods commonly used in the art, such as the protoplast method, competent cell method, electroporation method, and heat shock method, can be used.

In the second step of the method of the present invention, the host cells transformed above are cultured in an appropriate medium, and transformants that have been properly introduced are selected using marker gene expression, etc. as an indicator, and grown. This is the process of obtaining colonies. Although a vector library is used for transformation, only one type of vector is introduced into a single host cell, i.e., a toxic protein and one particular type of arbitrary peptide, or a toxic protein, a protease recognition sequence, and a certain type of vector. It is only a vector that expresses one specific type of arbitrary peptide. If this arbitrary peptide has the function of inhibiting the activity of the toxic protein, the synthesized toxic protein will be inactivated and will not exhibit toxicity to the host cells, resulting in the formation of colonies of transformants. . On the other hand, if this arbitrary peptide does not have such a function, the synthesized toxic protein exhibits activity, and its toxicity kills host cells and no colonies are formed. That is, this step is a step of screening for transformants expressing peptides that exhibit inhibitory activity against toxic proteins, based on determination of whether the host cells are alive or dead. The sequence of the inhibitory peptide can be determined, for example, by extracting DNA from cells that have grown and forming a colony according to a conventional method, using the DNA as a template, and amplifying a DNA fragment using appropriately set primers. It can be obtained by reading the sequence and converting the base sequence into an amino acid sequence. This step is also a step of expressing a toxic protein in host cells using a peptide that has the function of inhibiting the activity of the toxic protein.

The method for producing a toxic protein of the present invention further includes, as a third step, a step of isolating the toxic protein. This step is a step of culturing the obtained colony and isolating the expressed toxic protein from the culture. The medium and culture conditions used for colony culture can be appropriately selected by those skilled in the art depending on the type of host cell. Furthermore, the method for producing a toxic protein of the present invention may further include the step of purifying the isolated toxic protein. For isolation and/or purification of toxic proteins, methods common in the art, such as centrifugation, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., may be used alone or in appropriate combinations. good.
The isolated toxic protein is in a state where its activity is inhibited by the peptide. When a protease recognition sequence is linked to a toxic protein, an active form of the toxic protein can be obtained by cleaving the peptide with a protease that cleaves the recognition sequence. The presence or absence of cleavage of the peptide can be easily confirmed by SDS-PAGE or the like. Alternatively, when the peptide contains at least two Cys, the activated form of the toxic protein can be obtained by isolating the toxic protein from the culture based on the difference in reducing environment. This is because the intracellular environment is reducing, so Cys-containing peptides have a linear structure that inhibits the activity of toxic proteins, but by making the environment non-reducing after isolation, This method utilizes the fact that a disulfide bond is formed within a Cys-containing peptide, changing the folding of the peptide, and the peptide dissociates from the toxic protein and loses its inhibitory ability.

As shown in the Examples below, according to the method of the present invention, the toxic protein Piericin-1, specifically the N-terminal catalytic domain of Piericin-1 (shown at positions 1 to 233 of the amino acid sequence of SEQ ID NO: 4) It is possible to screen for peptides that inhibit the activity of a polypeptide consisting of an amino acid sequence), and also to express piericin-1 in host cells. Such an inhibitory peptide consists of the amino acid sequence represented by CLLMIRCLQFSVCGGSC (SEQ ID NO: 1), the amino acid sequence represented by GKNLNRSAPR (SEQ ID NO: 2), or the amino acid sequence represented by GHAEEYDDAAAARDSV (SEQ ID NO: 3), and pielysin-1 It is useful as an active ingredient in toxicity inhibitors. In the present invention, the toxicity inhibitor of piericin-1 is a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1, a peptide consisting of the amino acid sequence represented by SEQ ID NO: 2, and a peptide consisting of the amino acid sequence represented by SEQ ID NO: 3. In addition to these, pharmaceutical non-toxic carriers, stabilizers, wetting agents, emulsifiers, binders, isotonic agents, excipients, etc. may be included. It is also possible to appropriately add commonly used additives such as additives. In order to inhibit the toxicity of Piericin-1 using a Piericin-1 toxicity inhibitor, the inhibitor may be brought into contact with Piericin-1. The amount of the inhibitor to be used may be appropriately determined depending on the amount of Piericin-1 used, but for example, it is preferably 1 part by mass or more and 100 parts by mass or less as the inhibitory peptide per 1 part by mass of Piericin-1. More preferably 5 parts by mass or more and 50 parts by mass or less.

EXAMPLES Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1
Method (1) Preparation of DNA fragment containing Piericin-1 Using a plasmid that encoded the artificial gene of Piericin-1 having the base sequence of SEQ ID NO: 5, which was purchased from Eurofin Genomics Co., Ltd., the DNA fragment shown in Table 1 below was prepared. PCR was performed using two types of primer sets. The base sequence of SEQ ID NO: 5 is a sequence optimized for the piericin-1 gene. The forward primer contains a nucleotide sequence complementary to the antisense strand of the 5' terminal region of the piericin-1 gene, and the reverse primer contains a nucleotide sequence complementary to the nucleotide sequence encoding any peptide, a TEV protease recognition sequence ( It contains a sequence complementary to the nucleotide sequence encoding SEQ ID NO: 6) and a sequence complementary to the sense strand of the 3'-terminal region of the catalytic region of the piericin-1 gene. The arbitrary peptide sequence added by the C5x2 reverse primer is a sequence consisting of 17 amino acids in which the 1st, 7th, 13th, and 17th amino acids are Cys, and is added by the C6x2 reverse primer. The arbitrary peptide sequence is a sequence consisting of 19 amino acids in which the 1st, 8th, 15th, and 19th amino acids are Cys. For amino acids other than Cys at the above specific position in any peptide sequence, the codon NNK was used to reduce the probability of occurrence of a stop codon, so these reverse primers contain the base sequence MNN, which is the complementary strand of NNK. . By performing PCR using this primer set, a base sequence encoding a TEV protease recognition sequence and a base sequence encoding an arbitrary peptide are expressed downstream of the base sequence encoding the N-terminal catalytic domain of piericin-1. The operably linked DNA fragments were amplified. PCR was performed using KOD plus neo (manufactured by Toyobo Co., Ltd.) under the reaction solution composition shown in Table 2 and the reaction conditions shown in Table 3. After PCR, the reaction solution was subjected to agarose gel electrophoresis using a 1.5% agarose gel (Agarose LO3 "TAKARA", manufactured by Takara Bio Inc.). After electrophoresis, staining was performed using SYBR Green I Nucleic Acid Gel strain (manufactured by Lonza Japan Co., Ltd.), and the target band was cut out from the gel. DNA was extracted from the cut out gel using a QIAquick Gel extraction kit (manufactured by QIAGEN).

(2) Restriction enzyme treatment of vector and DNA fragment An expression vector was constructed using pETcoco-2 vector (manufactured by Merck Millipore). The pETcoco-2 vector is a protein expression vector that can control the copy number in E. coli after transformation. The copy number is maintained at about 1 copy in E. coli during cultivation in a normal medium, but it can be increased to about 40 copies by adding arabinose.
The pETcoco-2 vector and the C5×2 and C6×2 DNA fragments of pierisin-1 prepared in (1) were combined with NheI-HF and Hind III HF (both manufactured by New England Biolab Japan Co., Ltd.). ) was used for restriction enzyme treatment. After restriction enzyme treatment, the pETcoco-2 vector was subjected to electrophoresis using 0.8% agarose gel, and then the target band was excised from the gel, and DNA was extracted using QIAquick Gel extraction kit. The extracted pETcoco-2 vector was derived from E. Dephosphorylation treatment was performed using E. coli alkaline phosphotase (manufactured by Toyobo Co., Ltd.), and after the treatment, purification was performed using Nucleospin gDNA Clean-up (manufactured by Takara Bio Inc.). After restriction enzyme treatment, the DNA fragment was purified using Nucleospin gDNA Clean-up.

(3) Ligation reaction between vector and DNA fragment Two types of ligation reagents were used for the ligation reaction.
(i) Ligation using ligation high ver2 (manufactured by Toyobo Co., Ltd.) 2 μL of C5×2 and C6×2 DNA fragments prepared in (2), 8 μL of vector prepared in (2), and ligation high 10 μL of ver. 2 was mixed and reacted at 16° C. for 2 hours.
(ii) Ligation using ligation conventional kit (Nippon Gene) 2 μL of C5×2 and C6×2 DNA fragments prepared in (2), 8 μL of vector prepared in (2), and 10 μL of ligation conventional kit were mixed and reacted at 16°C for 10 minutes.

(4) Transformation of E. coli and cultivation of the obtained colony E. coli strain Tuner (DE3) pLysS (manufactured by Merck Millipore) was used as a host for protein expression. 1 μL of the ligation reaction solution prepared in (3) was added to 200 μL of Tuner (DE3) pLysS competent cells, and transformation was performed by a heat shock method at 42° C. for 45 seconds. After transformation, perform recovery culture using SOC medium at 37°C, centrifuge at 2300g for 10 minutes to collect bacterial cells, and culture overnight at 37°C on LB agar medium containing 50 μg/mL carbenicillin. went. Colonies formed after culturing were inoculated into LB liquid medium containing 50 μg/mL carbenicillin, and cultured overnight at 37°C. The next day, a glycerol stock of the obtained colony (strain) containing 16% glycerol was prepared using a portion of the culture solution, frozen in liquid nitrogen, and then stored at -80°C.

(5) Induction of protein expression Protein expression was performed using the bacterial solution prepared in (4). Inoculate the LB liquid medium containing 0.2% glucose and 50 μg/mL carbenicillin using the culture solution cultured in (4), and culture with shaking at 37°C until the turbidity OD600 becomes approximately 1.0. I did it. After the OD600 reached approximately 1.0, the culture solution was collected and centrifuged at 2300 g for 10 minutes. After centrifugation, discard the supernatant and suspend the pellet in 10 mL of LB liquid medium containing 50 μg/mL carbenicillin, then add IPTG to a final concentration of 0.1 mM and L-arabinose to a final concentration of 0.01%. Then, shaking culture was performed overnight at 20°C.

(6) Preparation of sample for cell disruption and expression analysis The culture solution prepared in (5) was centrifuged at 2300 g for 10 minutes. The supernatant was discarded, and the cells were suspended in 4 mL of a disruption buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 5% glycerol). After suspension, the bacterial cells were disrupted using ultrasound, and the disruption solution was used to prepare samples for SDS-PAGE. For sample preparation, Bolt LDS Sample Buffer (4x) and Bolt Sample Reducing Agent (10x) (both manufactured by Thermo Fisher Scientific KK) were used.

(7) Analysis by Western blotting Regarding the sample prepared in (6), the presence or absence of Piericin-1 synthesis was confirmed by Western blotting. Bolt 4-12% Bis-Tris Plus was used as the gel for SDS-PAGE, and NuPAGE MES SDS Running Buffer (20x) was diluted 20 times as the buffer for electrophoresis (both manufactured by Thermo Fisher Scientific K.K. ) . Anti-Piericin-1 rabbit antibody was used as the primary antibody, Anti-Rabbit IgG, HRP-Linked Whole Ab Donkey (manufactured by GE Healthcare Japan Co., Ltd.) was used as the secondary antibody, and Amarsham ECL Prime (GE Healthcare Japan Co., Ltd.) was used for color development.・Manufactured by Japan Co., Ltd.) was used. In the analysis, Piericin-1 composed of residues 1-233 synthesized in a cell-free synthesis system was used as a control.

(8) Sequence analysis of the peptide expressed in the strain in which the expression of piericin-1 was confirmed in (7). Sequence analysis was performed to identify the peptide sequence.
Of the culture solution prepared in (4), the remaining culture solution, excluding the glycerol stock and the portion used for inducing protein expression, was centrifuged at 2300 g for 10 minutes. The supernatant was discarded, and plasmid extraction was performed from the pellet using Qiaprep spin miniprep kit (manufactured by QIAGEN). PCR was performed using the obtained plasmid solution and the primers for sequence analysis shown in Table 4. The PCR reaction solution composition and reaction conditions were carried out under the conditions shown in Tables 2 and 3 (only the concentration of the plasmid solution was set to 0.26 μg/μL). After the reaction, ethanol precipitation was performed, and the pellet was dried and then dissolved in 50 μL of TE buffer to prepare a DNA solution. Using the prepared DNA solution, BigDye terminator ver3.1 Cycle Sequencing kit (manufactured by Thermo Fisher scientific K.K.) and 3500xL genetic analyzer (Thermo Sequencing was performed using Fisher scientific KK). For the analysis, the forward primer and reverse primer shown in Table 4 were used.

Results (1) Transformation Results As a result of the transformation experiment of method (4), the experiment was performed twice in a system using the ligation convenient kit with the C5x2 system, and a total of three colonies were obtained. In addition, the experiment was performed once in a system using the ligation high ver2 with the C6x2 system, and one colony was obtained. Of these, the colony obtained with the C5x2 system was cultured and used in the experiment in method (5).
When a gene encoding a protein that is not toxic to E. coli is introduced into E. coli as in this example, countless colonies can usually be obtained. However, the number of colonies obtained in this example was very small. Pierisin-1 has a very high cytotoxic activity, and it is expected that the sequences of peptides that inhibit this activity are limited. Most of the arbitrary peptides are peptides that do not exhibit inhibitory activity against Pierisin-1 or have moderate inhibitory activity, and since these peptides cannot completely suppress the activity of Pierisin-1, E. coli died. However, there were also very small amounts of peptides that exhibited strong inhibitory activity against Pierisin-1, and it is believed that these peptides allowed E. coli to survive and form colonies, although the number was very small.
Thus, in this example, very few peptides were obtained due to the activity of Pierisin-1, but this is also considered to indicate the high selection pressure and sensitivity of the method of the present invention.

(2) Results of expression analysis by Western blotting Figure 1 shows the results of Western blotting. Regarding the colonies obtained using the C5x2 system, a band with the same intensity as the control could be confirmed. From this, it was found that the colonies (strains) obtained using the C5x2 system expressed piericin-1. The band derived from the obtained strain was shifted toward the polymer side compared to the control band, which is because the number of amino acid residues in the control is 233, whereas in the obtained strain, the number of amino acid residues is 233. This is due to the fact that the number of amino acid residues is 273 because 30 residues including the protease recognition sequence and peptide sequence are added to the N-terminus, and 10 residues of the His tag sequence derived from the pETcoco-2 vector are added to the N-terminus. it is conceivable that.

(3) Sequence analysis results The results of the sequence analysis performed using method (8) are shown in FIGS. 2 and 3. FIG. 2 shows the sequencing results using the forward primer, and FIG. 3 shows the sequencing results using the reverse primer. In the figure, the part surrounded by a solid line is the sequence of piericin-1, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence. As a result of sequence analysis, the construct in which expression of Piericin-1 was confirmed had a Piericin-1 sequence, a protease recognition sequence, and a peptide sequence, and the sequence of the peptide was found to be CLLMIRCLQFSVCGGSC (SEQ ID NO: 1). Ta. Since piericin-1, which could not be expressed in E. coli using conventional methods, could be expressed, this peptide was considered to be a peptide that inhibits the toxicity of piericin-1.

Example 2
Method Expression analysis of piericin-1 was carried out in the same manner as methods (1) to (7) of Example 1, except that the primer set listed in Table 5 below was used to prepare a DNA fragment containing piericin-1. went. Note that the arbitrary peptide sequence added by the 15aa reverse primer is a peptide sequence consisting of 15 amino acids. In addition, peptide sequence analysis was performed in the same manner as method (8) of Example 1. However, only analysis from the reverse side was performed.

Results (1) Transformation Results As a result of the transformation experiment of method (4), the experiment was conducted once in a system using a ligation conventional kit, and three colonies were obtained. In addition, an experiment was conducted once using a system using ligation high ver. 2, and two colonies were obtained. Four of these colonies were cultured and used for the experiment in method (5).

(2) Results of expression analysis by Western blotting Figure 4 shows the results of Western blotting. It was possible to confirm the expression of piericin-1 in the four colonies (strains) obtained. Among these, strains 2 and 4 with strong expression were subjected to sequence analysis using method (8). In addition, the band derived from strain 4 was shifted toward the polymer side compared to the band of the control, but this is because the number of amino acid residues in the control is 233, whereas in strain 4, the number of amino acid residues is 233, whereas in strain 4, the C-terminus of piericin-1 This is due to the fact that the number of amino acid residues is 271 because 28 residues including the protease recognition sequence and peptide sequence are added to the N-terminus, and 10 residues of the His tag sequence derived from the pETcoco-2 vector are added to the N-terminus. it is conceivable that. On the other hand, the band derived from strain 2 was observed between the control band and the band derived from strain 4. Therefore, it is expected that a stop codon occurred in the peptide sequence, making the peptide sequence shorter than expected.

(3) Sequence analysis results The results of sequence analysis for strain 2 are shown in FIG. 5, and the results of sequence analysis for strain 4 are shown in FIG. In the figure, the part surrounded by a solid line is the piericin-1 sequence, the part surrounded by a broken line is the protease recognition sequence, and the part surrounded by a double line is the peptide sequence. As a result of sequence analysis, it was revealed that strains 2 and 4, in which piericin-1 expression was confirmed, both had constructs containing a piericin-1 sequence, a protease recognition sequence, and a peptide sequence. The peptide presented in strain 2 was a 10-residue peptide, 5 less than the designed one, due to the appearance of a stop codon in the sequence, and its sequence was GKNLNRSAPR (SEQ ID NO: 2). On the other hand, in strain 4, a peptide sequence of 15 residues as designed was added, and the sequence was GHAEEYDDAAAARDSV (SEQ ID NO: 3). Since piericin-1, which could not be expressed in E. coli using conventional methods, could be expressed, this peptide was considered to be a peptide that inhibits the toxicity of piericin-1.

Claims (5)

A plurality of vectors into which genes encoding fusion proteins containing a toxic protein and a peptide have been integrated, or a plurality of vectors into which genes encoding a fusion protein including a toxic protein, a protease recognition sequence, and a peptide have been integrated. transforming a host cell with a vector library comprising;
culturing the transformed host cells to obtain colonies;
culturing the obtained colony and isolating toxic proteins from the culture;
A method for producing a toxic protein, the method comprising:
The toxic protein is a protein that is toxic to the host cell, and the plurality of vectors are vectors having different base sequences encoding the peptide, provided that the peptide contains at least two cysteines. Production method. The manufacturing method according to claim 1, wherein the host cell is E. coli. The production method according to claim 1 or 2, wherein the peptide has a length of 3 to 50 amino acids. A gene encoding a fusion protein comprising Piericin-1 and a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, or a peptide consisting of the amino acid sequence of SEQ ID NO: 3, or Piericin-1, protease recognition host cells with a vector into which a gene encoding a fusion protein containing the sequence and the peptide consisting of the amino acid sequence of SEQ ID NO: 1, the peptide consisting of the amino acid sequence of SEQ ID NO: 2, or the peptide consisting of the amino acid sequence of SEQ ID NO: 3 is introduced. a step of transforming;
culturing the transformed host cells to obtain colonies;
culturing the obtained colony and isolating piericin-1 from the culture;
A method for producing piericin-1, comprising: Toxicity of Piericin-1 containing as an active ingredient one or more selected from the group consisting of a peptide consisting of the amino acid sequence of SEQ ID NO: 1, a peptide consisting of the amino acid sequence of SEQ ID NO: 2, and a peptide consisting of the amino acid sequence of SEQ ID NO: 3. inhibitor.