JP2017035063A - Method for selecting (poly) peptide/protein tag capable of covalently bonding to any target substance and (poly) peptide/protein tag selected and obtained - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for obtaining at high speed a (poly) peptide/protein tag for protein-labeling, which is covalently bonded to a target substance.SOLUTION: The present invention provides a method for selecting a peptide or a protein, which is capable of covalently bonding to any target substance. The method comprises the steps of: (a) preparing an RNA library by transcribing a DNA library; (b) preparing a library of RNA-linker-nucleic acid derivatives; (c) performing a s peptide or protein synthesis in a cell-free translation system to produce an mRNA display peptide/protein library; (d) reverse-transcribing an mRNA part to DNA to prepare a cDNA display peptide/protein library; (e) contacting the cDNA display peptide/protein library with an immobilized target substance; and (f) selecting a conjugate in which a peptide portion or a protein portion of the cDNA display peptide/protein library is covalently bonded to the target substance.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for selecting a (poly) peptide / protein tag capable of covalently binding to any target substance, (poly) peptide / protein tag, nucleic acid display peptide, nucleic acid display peptide / protein library, and covalent binding to a target substance The present invention relates to a method for obtaining a nucleic acid encoding a possible peptide / protein.

  In recent years, evolutionary molecular engineering has been developed and display technology has been put into practical use. For example, in phage display, a foreign protein is expressed as a fusion protein in a phage shell protein, a target protein that binds to a target substance is selected, and a gene encoding the target protein is analyzed.

  A protein that binds to a target substance obtained by such display technology is linked to the target protein and used as a mark (tag). (Poly) peptide / protein tag uses a specific target protein in a cell as a fluorescent molecule, etc. This method is useful in biological and medical research because it can be used for labeling and visualizing by the labeling and for controlling the activity and interaction of specific target proteins in cells.

(Poly) peptide / protein tags are already commercially available, for example, NEB “SNAP Tag”, Promega “Halo Tag”, Active motif “eDHFR Tag”, Invitrogen “Cys4 Tag” Etc.

  However, since the “SNAP Tag”, “Halo Tag”, and “eDHFR Tag” have a large tag size (molecular weight), the interaction between the target protein to which the (poly) peptide / protein tag is added and other biomolecules It tends to inhibit the intracellular localization. The “Cys4 Tag” includes an arsenic atom in a target substance that binds to a (poly) peptide / protein tag. Therefore, there is a problem that it is toxic and the selectivity of the binding between the (poly) peptide / protein tag and the target substance is relatively low.

  In addition, in the case of the (poly) peptide / protein tag selection method using cells as in the phage display, there is a drawback derived from the use of cells. For example, there are problems such as narrow detection space due to the use of cells, membrane permeability, bias by host cells, and limitation of libraries due to the number of host cells. Further, since the displayed peptide and the gene encoding it are not linked via a covalent bond, it cannot be used for selecting a (poly) peptide / protein tag by washing using a denaturant. Therefore, cell-free translation systems that do not use cells, such as ribosome display and mRNA display, have been developed.

For example, Patent Document 1 discloses a technique for producing a molecule in which a genotype and a phenotype are associated by binding a nucleic acid and a protein in a cell-free protein synthesis system.
If the genotype and the phenotype are associated with each other, when the target substance and the unknown protein of the phenotype are found to interact, the nucleic acid part of the genotype is amplified, thereby the unknown Proteins can be identified.

  Specifically, puromycin is linked to the 3 'end of mRNA via a spacer, and this ligated product is translated in a cell-free protein synthesis system to synthesize a protein. In the synthesis of the protein, puromycin on the 3 'end side of the mRNA is incorporated into the A site of the ribosome and is incorporated into the C terminus of the synthesized protein by the action of peptidyl transferase. As a result, it is possible to obtain a genotype-phenotype-corresponding molecule in which mRNA and a corresponding synthesized protein are linked via puromycin.

  However, since the displayed peptide (phenotype) and its encoding DNA (genotype) are not linked via a covalent bond, selection of (poly) peptide / protein tag by washing with a denaturant Cannot be used. In addition, it is a very time-consuming selection method because each of the multiple steps such as transcription, mRNA purification, puromycin modification, puromycin modification mRNA purification, translation, and mRNA display peptide purification must be performed individually. There was a problem.

Japanese Patent No.3683282

  The main object of the present technology is to provide a method for obtaining at a high speed a (poly) peptide / protein tag for protein labeling that is covalently bound to an arbitrary target substance.

  As a result of intensive studies to solve the above problems, the present inventors have found that the amino acid sequence of the (poly) peptide / protein tag is configured to be capable of covalently binding to a target substance, and the present invention is It came to be completed.

That is, the present technology includes the following steps:
(a) a step of transcribing a DNA library to prepare an RNA library;
(b) a step of linking a nucleic acid derivative via a linker to the 3 ′ end of RNA in an RNA library to produce an RNA-linker-nucleic acid derivative library;
(c) RNA-linker-nucleic acid derivative library, using the RNA portion of the library as mRNA, peptide or protein synthesis in a cell-free translation system to produce an mRNA display peptide / protein library,
(d) a step of reverse-trancribing the mRNA portion of the mRNA display peptide / protein library to DNA to produce a cDNA display peptide / protein library;
(e) contacting the immobilized target substance with a cDNA display peptide / protein library; and
(f) selecting a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library is covalently bound to the target substance;
And a method for selecting a peptide or protein that can be covalently bound to a target substance.

  The present technology also provides a peptide / protein tag containing a peptide or protein that can be covalently bound to a target substance, selected by the selection method.

  Furthermore, the present technology provides a nucleic acid display peptide / protein in which a peptide or protein capable of covalently binding to a target substance selected by the selection method and a nucleic acid encoding the peptide or protein are linked.

  In addition, the present technology provides a library composed of a plurality of nucleic acid display peptides / proteins having different nucleic acid sequences.

Furthermore, the present technology includes the following steps:
(a) a step of transcribing a DNA library to prepare an RNA library;
(b) a step of linking a nucleic acid derivative via a linker to the 3 ′ end of RNA in an RNA library to produce an RNA-linker-nucleic acid derivative library;
(c) RNA-linker-nucleic acid derivative library, using the RNA portion of the library as mRNA, peptide or protein synthesis in a cell-free translation system to produce an mRNA display peptide / protein library,
(d) a step of reverse-trancribing the mRNA portion of the mRNA display peptide / protein library to DNA to produce a cDNA display peptide / protein library;
(e) contacting the immobilized target substance with a cDNA display peptide / protein library;
(f) selecting a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library and the target substance are covalently bound, and
(g) A method for obtaining a nucleic acid encoding a peptide or protein capable of covalently binding to a target substance, comprising the step of amplifying cDNA of a selected conjugate.

Here, for example, the expression “RNA-linker-nucleic acid derivative” means that “linker” is linked to “RNA”, and further, “nucleic acid derivative” is linked to “linker”. Unless otherwise noted, other similar expressions are interpreted similarly.
For example, the expression “mRNA display peptide / protein library” means “mRNA display peptide library” or “mRNA display protein library”. Unless otherwise noted, other similar expressions are interpreted similarly.
For example, the expression “(poly) peptide / protein” consists of several amino acids, about 10 to several tens of amino acids, about 100 to several hundreds of amino acids. It means to include things.
Furthermore, for example, the expression “mRNA display peptide” means a molecule in which an mRNA fragment and a peptide translated from the fragment are associated with each other via a linker or a nucleic acid derivative. Unless otherwise noted, other similar expressions are interpreted similarly.

The present invention can provide a DNA display peptide / protein in which cDNA and a (poly) peptide / protein tag are irreversibly and stably bound by a covalent bond.
Note that the effects described here are not necessarily limited, and may be any of the effects described in the present specification.

It is a figure which shows the outline of the selection method of the (poly) peptide / protein which can be covalently bonded with the target substance of this invention. It is a figure which shows the outline of the selection method of polypeptide which concerns on embodiment of this invention. It is a figure which shows the outline of the preparation method of the cDNA display peptide library of the cell-free transcription / translation coupling system used by this invention. It is the graph which computed the ratio of the amount of collect | recovered cDNA with respect to all the prepared cDNA for every selection round. It is a figure which shows formation of a fluorescein modification pClBz-CRP tag-DHFR molecule | numerator. 2 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a CRP-DHFR tag fusion. FIG. 6 shows formation of DHFR-CRP tag-fluorescein modified pClBz molecules. 2 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a DHFR-CRP tag fusion. FIG. 5 shows formation of fluorescein modified pClBz-CRP tag-protein molecules. FIG. 5 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a CRP tag-protein fusion. It is a figure which shows formation of a fluorescence label molecule modification pClBz-polypeptide tag-DHFR molecule. FIG. 6 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a CRP tag-DHFR fusion. FIG. 5 shows formation of a fluorescein modified pClBz-CRP fragment tag-DHFR molecule. FIG. 6 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a CRP fragment tag-DHFR fusion. It is the graph which computed the ratio of the amount of collect | recovered cDNA with respect to all the prepared cDNA for every selection round. FIG. 6 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a CRP tag-DHFR fusion. FIG. 5 shows formation of a fluorescein modified polypeptide tag-DHFR molecule. FIG. 5 is a drawing-substituting photograph of an SDS-PAGE electrophoresis image of a fluorescein-modified polypeptide tag-DHFR molecule. It is a figure which shows formation of a NLS-GFP-CRP tag-label molecule modified pClBz molecule. It is a drawing-substituting photograph of a microscopic image of a cell expressing a fluorescently labeled polypeptide tag fusion protein.

The method for selecting a (poly) peptide or protein capable of covalently binding to a target substance of the present technology includes the following steps (a) to (f), and may further include a step (g).
(a) a step of transcribing a DNA library to prepare an RNA library;
(b) a step of linking a nucleic acid derivative via a linker to the 3 ′ end of RNA in an RNA library to produce an RNA-linker-nucleic acid derivative library;
(c) RNA-linker-nucleic acid derivative library, using the RNA portion of the library as mRNA, peptide or protein synthesis in a cell-free translation system to produce an mRNA display peptide / protein library,
(d) a step of reverse-trancribing the mRNA portion of the mRNA display peptide / protein library to DNA to produce a cDNA display peptide / protein library;
(e) contacting the immobilized target substance with a cDNA display peptide / protein library;
(f) selecting a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library and the target substance are covalently bound, and
(g) Amplifying the cDNA of the selected conjugate.

  The steps are performed in the order of steps (a) to (g). Steps (a) to (c) automatically proceed continuously. Further, the process up to step (e) can be carried out in one pot without going through purification. Furthermore, by repeating the steps (a) to (g), the target (poly) peptide or target protein that interacts with the target substance (the (poly) peptide / protein tag to be obtained) can be concentrated at high speed.

The large amount of cDNA amplified in step (g) is regarded as a new DNA library, transcribed in the next step (a) to prepare an RNA library, and the steps after step (b) are performed. A DNA encoding a target (poly) peptide or target protein (a (poly) peptide / protein tag to be obtained) that rapidly interacts with a target substance can be selected. For example, even if the initial DNA library used in the first step (a) of the present technology contains about 10 ¹⁵ kinds of polypeptides, it can be screened at high speed.

According to this technology, a selection method using mutagenesis, a selection method by individual screening (up to about 10 ⁵ types of polypeptides can be screened), a phage display method or a yeast display method (up to about 10 ⁹ types of polypeptides) (Poly) peptide / protein tags can be selected at a high speed from a very large (about 10 ¹⁵ kinds) library.

  Further, after the steps (a) to (g), a step (h): a step of determining the sequence of the amplified cDNA can be performed. By this step, the target peptide or target protein ((poly) peptide / protein tag to be obtained) that interacts with the target substance can be identified. The flow of steps (a) to (g) and (h) is shown in FIG.

The gene manipulation techniques such as library preparation, transcription, cell-free translation, reverse transcription, and amplification performed in the steps (a) to (h) can be performed by known methods. For example, Sambrook et al. (1989) Molecular Cloning, 2 nd Edition, can be carried out according to the method described in Cold Spring Harbor Laboratory Press.

  In step (a), an RNA library is prepared from the DNA library by transcription, and then the process proceeds to step (b). Examples of the linker used in step (b) include polymers that can be linked to the 3 ′ end of RNA, such as natural or synthetic nucleic acid chain fragments, polysaccharides, polyethylene glycol, and DNA chain / polyethylene glycol chimeras. . The length of the linker is, for example, about 100 to 1000 mm, but is not limited as long as the effect of the present invention is not hindered.

The 3 'end of RNA is linked to one end of the linker, and the nucleic acid derivative is linked to the other end. A nucleic acid derivative is a substance having a chemical structure skeleton similar to a nucleoside or nucleoside.
Nucleic acid derivatives include, for example, puromycin, 3'-N-aminoacylpuromycin aminonucleoside (PANS-amino acid), the amino acid part of which is glycine PANS-Gly, valine PANS-Val, alanine PANS-Ala, etc. , PANS-all amino acids corresponding to all amino acids, or 3'-N-aminoacyl adenosine aminonucleoside (AANS-amino acid), the amino acid part of which is glycine AANS-Gly, valine AANS-Val, alanine AANS-Ala, etc. AANS-all amino acids corresponding to all amino acids.

Next, (poly) peptide or protein synthesis is performed in a cell-free translation system using the RNA portion of the RNA-linker-nucleic acid derivative library prepared in step (b) as mRNA (step (c)).
The nucleic acid derivative portion of the RNA-linker-nucleic acid derivative is incorporated into the ribosome and incorporated into the C-terminus of the translated (poly) peptide or protein as a substrate for the peptide transfer reaction. As a result, the 3 ′ end of mRNA is linked to (poly) peptide or protein via a linker-nucleic acid derivative, and mRNA-linker-nucleic acid derivative- (poly) peptide / protein conjugate, ie, mRNA display peptide / Protein is formed.

  Next, the step (d) of reversely transcribing the mRNA portion of the mRNA display peptide / protein library prepared in the step (c) to cDNA is performed. This forms a cDNA display peptide / protein library.

Next, before the step (e) is performed, the target substance is immobilized on a solid phase in advance. The solid phase is not particularly limited as long as it is generally used as a solid phase carrier such as magnetic beads or affinity beads.
A known technique may be used as the immobilization method. In addition, after immobilizing the target substance, it is preferable to treat the solid phase surface so that substances other than the target substance do not bind to the solid phase carrier.
In step (e), the immobilized target substance is brought into contact with the cDNA display peptide / protein library.

  When the cDNA display peptide / protein library is brought into contact with the immobilized target substance, the target substance specifically binds to the peptide part or the protein part. The target substance bound non-specifically to the cDNA display peptide / protein, weakly bound (not covalently bound), or bound to the solid support can be removed by washing under strong conditions.

The strong washing condition is, for example, washing with an organic solvent such as DMSO or SDS, a denaturing agent, etc., and those that do not affect the amplification function of cDNA as much as possible are preferable.
Specifically, there is no particular problem if it is washed with an organic solvent, a denaturant, or washed under heating conditions up to 95 ° C., which is also used for PCR conditions.

  By the washing, a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library is covalently bound to the target substance is selected (step (f)).

  Since the covalently bound target substance-cDNA display peptide / protein remains under strong washing conditions, its cDNA portion is amplified (step (g)). The amplification product is applied to step (a) as a DNA library without purification as it is, enters the next selection round, and steps (b) to (g) are performed. By repeating this, a (poly) peptide tag or protein tag that binds to the target substance is selected.

  After amplifying the cDNA portion of the cDNA display peptide / protein having a (poly) peptide / protein tag that binds to the target substance in step (g), the sequence of the cDNA is determined (step (h)). Thereby, (poly) peptide / protein interacting with the target substance can be analyzed. The identified (poly) peptide or protein can be used not only as a (poly) peptide tag or protein tag, but also for research of interaction with drugs, development of pharmaceuticals, and the like.

<Embodiment 1>
In this embodiment, p- (chloromethyl) benzamide (pClBz) is described as an example of a small molecule (target small molecule) of a target substance.
pClBz is a small molecule of the size represented by the molecular formula C ₉ H ₁₀ ClNO and can be introduced into the cytoplasm of living cells.

In order to obtain a (poly) peptide / protein tag that reacts with pClBz, a DNA library is prepared, and a peptide tag library is prepared through transcription and translation from the DNA library. The peptide tag library is designed so that an amino acid sequence consisting of 8 to 15 random amino acids is inserted between the N-terminal formylmethionine and the C-terminal Gly ₅ -Ser spacer.

  Specifically, an mRNA library transcribed from a DNA library is applied to a cell-free translation system. At this time, when a DNA chain is used as a linker, it is modified with puromycin and translated, an mRNA display peptide having the structure of mRNA-DNA linker-puromycin-peptide is formed.

  Next, reverse transcription of the mRNA portion of the mRNA display peptide yields a cDNA display peptide library. Here, the step of purifying the cDNA display peptide library is not necessary.

Subsequently, the target small molecule pClBz immobilized on the magnetic beads is mixed with an unpurified cDNA display peptide library. The Cl portion of the molecular formula C ₉ H ₁₀ ClNO of the target small molecule pClBz reacts with its thiol group if the peptide portion of the cDNA display peptide contains, for example, a cysteine residue. This selects peptides that react with pClBz. In this operation, in order to make all thiol groups free, selection is performed under the conditions of pH 7.6, 37 ° C., 60 minutes in the presence of the reducing agent tris (2-carboxyethyl) phosphine (TCEP).

To remove (poly) peptide / protein tags that did not bind to the pClBz immobilized on the magnetic beads, the beads were washed, and the cDNA corresponding to the selected (poly) peptide / protein was amplified by PCR and amplified. The product is used as a library to select the next (poly) peptide / protein.
Thus, the first (poly) peptide / protein selection round is completed.
The series of steps using the target small molecule pClBz of this embodiment is shown in FIG.

Subsequently, in the second round of (poly) peptide / protein selection, the amplification product cDNA library is subjected to a cell-free transcription / translation coupling system. At this time, puromycin-DNA linker for cDNA display is added as described above. Here, an mRNA display peptide library is formed by transcription, and a cDNA display peptide library is formed by subsequent translation.
An outline of a method for preparing a cDNA display peptide library of the cell-free transcription / translation coupling system is shown in FIG.

  For unpurified cDNA display peptide libraries, clearance is performed over multiple selection rounds using magnetic beads that are not bound to pClBz to remove (poly) peptides / proteins that bind non-specifically to the beads. It is preferable.

Furthermore, after reacting the pClBz-immobilized magnetic beads with the cDNA display peptide, the (poly) peptide in which pClBz and magnetic beads are non-covalently bonded while selecting the (poly) peptide / protein tag covalently bonded to pClBz. / Wash with denaturing reagents (urea and SDS) and organic solvent (DMSO) to remove protein tags. In the present technology, the target small molecule and the (poly) peptide / protein tag are strongly bonded by a covalent bond, and thus can be washed under strong conditions.
Also, in order to remove (poly) peptide / protein tags that recognize both the pClBz molecule and the magnetic bead portion, two types of magnetic beads can be used alternately in multiple selection rounds as magnetic beads to immobilize pClBz. preferable.

For the amount of cDNA recovered in the selection round, the ratio to the total amount of cDNA prepared using quantitative PCR is calculated. An example is shown in the graph of FIG. As the round progresses, reducing the reaction time from 60 minutes to 3 minutes or reducing the amount of magnetic beads with immobilized pClBz increases the selection pressure.
Further, for example, the cDNA collected in rounds 11 to 13 in FIG. 4 can be subjected to error-prone PCR amplification to carry out random mutagenesis to enhance the reactivity of the (poly) peptide / protein tag.

In this embodiment, the following two (poly) peptide / protein tags that react with the target small molecule pClBz are selected.
Polypeptide tag CRP1: THWFWCPYWGWLRS (SEQ ID NO: 1)
Polypeptide tag CRP2: WFWCPYWRTYIWY (SEQ ID NO: 2)
In CRP1, the sixth amino acid is cysteine, and in CRP2, the fourth amino acid is cysteine.
These polypeptide tags have a common motif WFWCPYW (SEQ ID NO: 3).

The two polypeptide tags CRP1 and CRP2 are expressed using a cell-free transcription / translation coupling system in the absence of DNA linker-puromycin. It is then incubated with p- (chloromethyl) -N- (2-hydroxyethyl) benzamide for 30 minutes at 37 ° C.
When this is analyzed by MALDI-TOF-MS, it can be seen that the two kinds of polypeptide tags are covalently bonded to p- (chloromethyl) -N- (2-hydroxyethyl) benzamide.
When the polypeptide tag is expressed so that a cysteine residue is added to the C-terminus of the polypeptide tag and treated with p- (chloromethyl) -N- (2-hydroxyethyl) benzamide, p- (chloro Those that react with one methyl) -N- (2-hydroxyethyl) benzamide are detected, but those that react with two or more are not detected.
Furthermore, when the cysteine residue at the C-terminal of the polypeptide tag is changed to a serine residue and treated with p- (chloromethyl) -N- (2-hydroxyethyl) benzamide, the polypeptide tag does not react at all.
That is, the two kinds of polypeptide tags are labeled with p- (chloromethyl) -N- (2-hydroxyethyl) benzamide via a cysteine residue in a site-specific manner.

  In order to investigate the labeling specificity of the two types of polypeptide tags CRP1 and CRP2, a fusion of the polypeptide tag and DHFR (dihydrofolate reductase) is expressed in vitro (DHFR-CRP tag), Treat with fluorescein modified pClBz for 30 minutes at 37 ° C. Then, a molecule having a structure shown in FIG. 5 (fluorescein modified pClBz-CRP-DHFR tag) is formed.

When the formed product is subjected to SDS-PAGE electrophoresis, it is shown in FIG. 6 (left: Coomassie Brilliand Blue, right: in-gel fluorescence). Lane 1 is a negative control containing only DHFR and no polypeptide tag. Lane 2 is the CRP1-DHFR fusion described in Embodiment 1, and lane 3 is the CRP2-DHFR fusion.
From this figure, the polypeptide tag-DHFR fusion is specifically labeled with fluorescein-modified pClBz. This indicates that fluorescein-modified pClBz binds to the thiol group of the cysteine residue contained in the polypeptide tag.

  In addition, a fusion (DHFR-CRP tag, FIG. 7) in which CRP1 or CRP2 is fused to the C terminus of DHFR can be specifically modified by treating it similarly with fluorescein-modified pClBz (FIG. 8).

  Furthermore, other proteins such as TNF (Tumor Necrosis Factor) or PPIA (peptidylprolyl isomerase A, peptidylpropyl isomerase A) and the aforementioned CRP1 or CRP2 in place of DHFR (FIG. 9) are also fluorescein. It can be specifically labeled with modified pClBz (Figure 10).

  Alternatively, instead of fluorescein, pClBz modified with another label such as coumarin, Alexa Fluoro 488 (AF488) or tetramethylrhodamine (TMR) can be prepared and treated in the same way (FIG. 11). It can be labeled (Figure 12).

The preferred length of the (poly) peptide tag for efficient labeling is that the (poly) peptide tag of various lengths obtained by cleaving CRP1 or CRP2 is fused to DHFR to express the fusion, and the same as described above. It can be examined by labeling with fluorescein modified pClBz under conditions.
For example, a (poly) peptide tag containing WFWCPYW (SEQ ID NO: 3), which is a common motif of CRP1 (SEQ ID NO: 1) and CRP2 (SEQ ID NO: 2), but having a different length is prepared (FIGS. 13 and 14)) .
When this is analyzed by SDS-PAGE, it is shown that CRP1 and CRP2 closer to the full length than those obtained by shortening CRP1 and CRP2 are efficiently labeled (electrophoresis image in FIG. 14).

Next, the acquisition of (poly) peptide / protein tags with higher reactivity will be examined.
There are 5 random amino acids between the N-terminal fMet-Ser-Gly ₂ (SEQ ID NO: 4) of the (poly) peptide / protein tag library and the common motif WFWCPYW (SEQ ID NO: 3), and A (poly) peptide / protein tag library is created with 5 random amino acids between the common motif and the C-terminal Gly ₅ -Ser (SEQ ID NO: 5) spacer.
For the (poly) peptide / protein tag library, after repeating the above selection round 10 times, when sequencing, 5 random amino acids on the N-terminal side do not converge, but 5 random amino acids on the C-terminal side Converges mainly to the CCWVP sequence (SEQ ID NO: 6) (FIG. 15).
This is the polypeptide tag CRP3 (WFWCPYWCCWVP, SEQ ID NO: 7), and in the same manner as described above, the efficiency when CRP3 and DHFR are fused and labeled with fluorescein is comparable to that of CRP1 (FIG. 16). .

Next, the irreversibility of the label due to the covalent bond is examined.
For example, a fluorescein-labeled polypeptide tag (CRP) -fused DHFR and a tetracysteine peptide tag (Cys ₄ , SEQ ID NO: 8) -fused DHFR that binds to the red biarsenical dye (ReAsH) commercially available from Invitrogen 17) is treated with 2-mercaptoethanol (BME) at 95 ° C. for 5 minutes.
When the treated product is analyzed by SDS-PAGE, the fluorescein (FlAsH) intensity of red biarsenical dye-bound tetracysteine peptide tag fusion DHFR decreases after 2-mercaptoethanol treatment. On the other hand, the fluorescein intensity of fluorescein-labeled polypeptide tag (CRP) -fused DHFR is constant even after treatment (FIG. 18). That is, it shows the irreversibility of labeling of a polypeptide tag (CRP) containing cysteine. This is considered to be advantageous even under strong washing conditions, for example, when intracellular proteins are labeled and washed.

To check whether the (poly) peptide / protein tag fusion protein is labeled inside the living cell, connect GFP to the nuclear localization sequence (NLS) and add CRP1-2 to the N-terminus of GFP. Design each fusion (Figure 19). Here, the nuclear localization sequence (NLS) is (DPKKKRKV) ₃ (SEQ ID NO: 9).
The designed NLS-GFP-polypeptide tag (CRP1-2) fusion is expressed in human embryonic kidney (HEK) cells. On the other hand, instead of CRP1-2, a fusion with a tetracysteine peptide tag is also expressed.
Next, for example, 0.1 μM membrane-permeable TMR (tetramethylrhodamine) modified pClBz (TMR-pClBz) or ReAsH is labeled by incubating at 37 ° C. for 30 minutes.
In transfected cells (identified with GFP), nuclei are labeled with TMR, whereas untransfected cells are not labeled. CRP1-2 is labeled in the same manner as the Cys ₄ tag labeled with control ReAsH.
Looking at the microscopic image, in the left column of FIG. 20, the NLS-GFP-polypeptide tag (CRP1-2) emits green light, indicating the presence of transfected cells. In the center column of FIG. 20, TMR-pClBz binds to peptide tags CRP1-2, or ReASH binds to a tetracysteine peptide, indicating that it emits red light. The right column of FIG. 20 shows a composite image of the green fluorescence image and the differential interference microscope image.

Hereinafter, the present invention will be described in more detail based on examples. In addition, the Example demonstrated below shows an example of the typical Example of this invention, and, thereby, the range of this invention is not interpreted narrowly.
The details of the primers, DNA encoding (poly) peptide / protein, reagents, etc. used in this example will be described after this example.

[1. Selection of (poly) peptide / protein tag that reacts with pClBz (target substance)]
SD8M2-NNK8-15-G5.F60 oligo DNA (SEQ ID NO: 10) and G5S-4an21.R41 oligo DNA (SEQ ID NO: 11) are annealed to prepare a random (poly) peptide / protein-encoding DNA template. , Primer extension with KOD-plus-neo DNA polymerase (TOYOBO), and then the obtained double-stranded DNA was converted into T7SD8M2.F44 primer (SEQ ID NO: 12) and G5S-4an21.R41 primer (SEQ ID NO: 13). Was amplified with Taq DNA polymerase (Genscript). The amplified DNA was purified by extraction with phenol / chloroform and ethanol precipitation. The random DNA template thus prepared was transcribed by run-off in vitro transcription using T7 RNA polymerase (TOYOBO), and the random mRNA was purified by isopropanol precipitation. The mRNA library was prepared by mixing random mRNA containing 8 to 15 repetitive NNK sequences. A library of mRNA display peptides was prepared by translating with a release factor-free translation system (PURExpress ΔRF123 Kit, New England Biolabs). At this time, the measurement was performed at 37 ° C. for 25 minutes on a 300 μL scale containing 2.5 μM mRNA library and 2.5 μM puromycin linker (BEX). EDTA was then added to the mRNA display peptide library solution to dissociate the ribosome. Reverse transcription of mRNA display peptide library using RNase H-inactivated reverse transcriptase (ReverTraAce, TOYOBO) and G5S-4.R20 primer (SEQ ID NO: 14) at 42 ° C before performing the selection procedure. Went for a minute.

The reverse transcription reaction is quenched with EDTA, neutralized with HEPES solution, all mercapto groups are reduced with tris (2-carboxyethyl) phosphine (TCEP), and p- (chloromethyl) immobilized on magnetic beads Benzamide (pClBz) (Dynabeads, Life Technologies) was mixed with the peptide library solution at 37 ° C. for 60 minutes. After removing the supernatant, the magnetic beads are washed three times with HBS-T (50 mM HEPES-K pH 7.5, 300 mM NaCl, 0.05% tween 20) to encode the cDNA bound to the pClBz-immobilized beads (poly) Peptides / proteins were added to the PCR pre-mixture solution (10 mM Tris-HCl pH 8.4, 50 mM KCl, 0.1% (v / v) Triton X-100, ₂ mM MgCl ₂ , 0.25 mM each dNTP, 0.25 μM T7SD8M2. Diluted with F44 (SEQ ID NO: 12), 0.25 μM G5S-4an21.R41 (SEQ ID NO: 11), the selected cDNA bound to the magnetic beads was eluted at 95 ° C. for 5 minutes. The amount was quantified by quantitative PCR (StepOnePlus, Applied Biosystems) using SYBR, using T7SD8M2.F44 (SEQ ID NO: 12) and G5S-4an21.R41 (SEQ ID NO: 11) as primers. PCR amplification using Taq DNA polymerase (Genscript) was performed for the next selection round.

  From the second round, a release factor-free transcription / translation-coupling system (PURExpress ΔRF123 Kit, New England Biolabs) containing an unpurified PCR mixture containing puromycin linker (BEX) and the cDNA library from the previous step. ) Was transcribed and translated to prepare an mRNA display peptide library. The prepared non-covalent mRNA display peptide library was reverse transcribed using ReverTraAce at 42 ° C. for 30 minutes to obtain a covalently bound cDNA display peptide library. The reverse transcription reaction was quenched with EDTA, neutralized with HEPES solution, all mercapto groups were reduced with TCEP, and the cDNA display peptide library was combined with magnetic beads without pClBz (Dynabeads, Life Technologies) at 25 ° C. Alternatively, it was incubated for negative selection at 37 ° C. for 5 minutes. The negative selection was performed 5 times to remove (poly) peptide / protein bound to the magnetic beads. Subsequently, the supernatant was mixed with pClBz-immobilized beads (Dynabeads, Life Technologies or FG beads, Tamagawa seiki) at 37 ° C. for positive selection, and DMSO, SDS-containing and pClBz-containing HBS-T, 37 Washed 5 times at ° C. The selected cDNA bound to the beads is quantified by quantitative PCR (StepOnePlus, Applied Biosystems) using SYBR, using T7SD8M2.F44 (SEQ ID NO: 12) and G5S-4an21.R41 (SEQ ID NO: 11) as primers. Amplified with PCR Taq DNA polymerase for the next round of selection.

[2. Labeling of in vitro expressed CRP fusion polypeptide and DHFR with pClBz-modified small molecule by cell-free translation system]
CRP fusion polypeptide and DHFR can be transcribed and translated in vitro at 37 ° C for 60 minutes using a transcription / translation-coupling system (PURExpress, New England Biolabs) containing the corresponding PCR amplified DNA template. Prepared. Label CRP fusion polypeptide and DHFR with 10 μM p- (chloromethyl) -N- (2-hydroxyethyl) benzamide or fluorescein-pClBz in the presence of 1 mM TCEP at 37 ° C. for 30 minutes did. The sample was desalted using C18-tip (C-TIP, Nikkyo Technos) and eluted with 0.5% acetic acid and 80% acetonitrile saturated with α-cyano-4-hydroxycinnamic acid (CHCA). MALDI-TOF -MS (AXIMA-CFR, SHIMADZU) analysis was performed in linear positive mode. For SDS-PAGE analysis, samples were run on 10% SDS-PAGE and visualized with Coomassie brilliant blue staining and in-gel fluorescence (ImageQuant 400, GE Healthcare).

[3. Labeling of CRP fusion protein with TMR-pClBz in living cells]
Human embryonic kidney (HEK) cells are cultured using collagen type I-coated chamber cover glass (IWAKI) and nuclear-localized polypeptide tag-fused GFP (CRP-GFP-NLS) using Lipofectamine 2000 (Life Technologies) Or Cys4-GFP-NLS). The cells were then cultured for 36 hours in medium at 37 ° C. with 5% CO ₂ . To wash away excess TMR-pClBz or ReAsH, cells were rinsed 3 times with media and incubated at 37 ° C. for 6 hours. After washing the cells with DPBS, the cells were observed alive with an epifluorescence microscope (IX71, Olympus).

[Details of primers, (poly) peptide / protein-encoding DNA, reagents, etc.]
(1. Chemical synthesis of p- (chloromethyl) benzoic acid cyanomethyl ester)
p- (Chloromethyl) benzoic acid cyanomethyl ester is described in Kawakami, T., Ishizawa, T. & Murakami, H. Extensive reprogramming of the genetic code for genetically encoded synthesis of highly N-alkylated polycyclic peptidomimetics. Journal of the Synthesized as described in American Chemical Society 135, 12297-12304 (2013).

(2. Preparation of p- (chloromethyl) benzoyl-tRNA ^fme t (CAU))
E. coli initiator tRNA ^fMet (CAU) was prepared by run-off in vitro transcription using T7 RNA polymerase as described in Kawakami et al. P- (chloromethyl) benzoyl-tRNAfmet (CAU) was also prepared according to the procedure of Kawakami et al.

(3. Preparation of DNA encoding peptide)
The primers used to prepare the template DNA are shown in the table below. DNA encoding the peptide was appropriately prepared with Taq DNA polymerase (Genscript) using a forward primer and a reverse primer suitable for primer extension and a forward primer and a reverse primer suitable for PCR amplification.

(4. Preparation of DNA encoding CRP fusion (poly) peptide / protein)
A linear DNA encoding a CRP fusion (poly) peptide / protein for in vitro translation using the primers shown in Table 1 above, and KOD-plus- using a forward primer and a reverse primer suitable for PCR amplification. Prepared with neo DNA polymerase (TOYOBO). In PCR amplification of DHFR gene, PURExpress DHFR control template (New England Biolabs) was used as template DNA. Human cDNA clones were used as template DNA in PCR amplification of tumor necrosis factor-α (TNF) and peptidyl-propyl cis-trans isomerase (PPIA) genes. In order to prepare plasmid DNA for transfection, the polypeptide tag CRP was introduced into the sticky end by annealing a complementary oligo DNA encoding CRP. The annealed insert was introduced into plasmid DNA that had been treated with the appropriate restriction enzymes.

(5. Preparation of p- (chloromethyl) benzamide-immobilized beads)
Amine magnetic beads (Dynabeads, Life Technologies or FG beads, Tamagawa seiki) were washed twice with 273 mM diisopropylethyleneamine (DIEA) in dimethylformamide (DMF), and in the presence of 273 mM DIEA in DMF for 60 minutes. , P- (chloromethyl) benzoyl chloride. The beads were washed 3 times with 10 mM DIEA in DMF and washed 3 times with 10 mM Tris / HCl (pH 6.8) containing 16.6% DMF.

(6. Cell culture and transfection)
HEK cells were cultured at 37 ° C. under 5% CO ₂ using DMEM (Wako) containing 10% (v / v) fetal bovine serum (FBS, GIBCO). Lipofectamine 2000 was used to transfect up to 70% confluent cells.

(7. Labeling with in vitro expressed CRP fusion (poly) peptide / protein and DHFR with pClBz-modified small molecule in cell-free translation system)
CRP fusion (poly) peptide / protein can be transcribed and translated in vitro at 37 ° C for 60 minutes using a transcription / translation-coupling system (PURExpress, New England Biolabs) containing the corresponding PCR amplified DNA template. Prepared to go. CRP fusion peptides and proteins were used in the presence of 1 mM TCEP using 10 μM p- (chloromethyl) -N- (2-hydroxyethyl) benzamide, fluorescein-pClBz, coumarin-pClBz, AF488-pClBz or TMR-pClBz And labeling at 37 ° C. for 30 minutes. The sample was desalted using C18-tip (C-TIP, Nikkyo Technos) and eluted with 0.5% acetic acid and 80% acetonitrile saturated with α-cyano-4-hydroxycinnamic acid (CHCA). MALDI-TOF -MS (AXIMA-CFR, SHIMADZU) analysis was performed in linear positive mode. For SDS-PAGE analysis, samples were denatured with 10% SDS-PAGE and visualized with Coomassie brilliant blue staining and in-gel fluorescence (ImageQuant 400, GE Healthcare). Cys4 fusion DHFR was labeled in the same manner using FlAsH instead of fluorescein-pClBz.

(8. pClBz-reactive (poly) peptide / protein tag sequence optimization based on the DIVERSE system)
To prepare the DNA library, MSG2-NNK5-WFWCPYW-NNK5-G5S.F87 oligo DNA (SEQ ID NO: 17) and newG5S.an21.R41 oligo DNA (SEQ ID NO: 18) were annealed and KOD-plus-neo DNA polymerase (TOYOBO) primer extension, and then the resulting double-stranded DNA was subjected to Taq DNA polymerase using T7SD8M2-Sgcc2.F53 primer (SEQ ID NO: 19) and newG5S.an21.R41 primer (SEQ ID NO: 18). Amplified with (Genscript). The amplified DNA was extracted with phenol / chloroform and ethanol precipitation. The prepared DNA library was transcribed by run-off in vitro transcription using T7 RNA polymerase (TOYOBO), and the mRNA library was purified by isopropanol precipitation. The library of mRNA display peptides was released at 37 ° C on a 250 μL scale containing 2.5 μM mRNA library and 2.5 μM puromycin linker (BEX) using the release factor-free translation system (PURExpress ΔRF123 Kit, New England Biolabs). And by translating under 25 min conditions. Next, EDTA was added to the mRNA display peptide library to dissociate the ribosome. Reverse transcription of mRNA display peptide library using RNase H-inactivated reverse transcriptase (ReverTraAce, TOYOBO) and newG5S.R20 primer (SEQ ID NO: 20) under the condition of 42 ° C. for 30 minutes before the selection round I went there.

After quenching the reverse transcription reaction with EDTA, the solution was neutralized with HEPES, all thiol groups were reduced with tris (2-carboxyethyl) phosphine (TCEP), and immobilized on magnetic beads (Dynabeads, Life Technologies) p- (Chloromethyl) benzamide (pClBz) was mixed with the peptide library solution at 37 ° C. for 10 minutes. After removing the supernatant, the beads were washed 3 times with HBS-T (50 mM HEPES-K pH 7.5, 300 mM NaCl, 0.05% tween 20), and the peptide-encoding cDNA bound to the pClBz-immobilized beads was removed. , PCR pre-mixture solution (10 mM Tris-HCl pH 8.4, 50 mM KCl, 0.1% Triton X-100, ₂ mM MgCl ₂ , each 0.25 mM dNTP, 0.25 μM T7SD8M2-Sgcc2.F53 (SEQ ID NO: 19), 0.25 Diluted with μM G5S-4an21.R41 (SEQ ID NO: 11)), the selected cDNA on the beads was eluted at 95 ° C. for 5 minutes. The amount of cDNA eluted was quantified using SYBR green quantitative PCR (StepOnePlus, Applied Biosystems) using T7SD8M2-Sgcc2.F53 (SEQ ID NO: 19) and G5S-4an21.R41 (SEQ ID NO: 11) as primers. did. The eluted cDNA was PCR amplified with Taq DNA polymerase (Genscript) and subjected to the next selection round.

  From the second round, a release factor-free transcription / translation-coupling system (PURExpress ΔRF123 Kit, New England Biolabs) containing an unpurified PCR mixture containing puromycin linker (BEX) and the cDNA library from the previous step. ) Was transcribed and translated to prepare an mRNA display peptide library. The prepared non-covalent mRNA display peptide library was reverse transcribed using ReverTraAce at 42 ° C. for 30 minutes to obtain a covalently bound cDNA display peptide library. The reverse transcription reaction was quenched with EDTA, neutralized with HEPES solution, all thiol groups were reduced with TCEP, and the cDNA display peptide library was added to pClBz-free magnetic beads (Dynabeads, Life Technologies) at 25 ° C. Alternatively, it was incubated for negative selection at 37 ° C. for 5 minutes. The negative selection was performed 5 times to remove peptides that bound to the magnetic beads. Subsequently, the supernatant was mixed with pClBz-immobilized beads (Dynabeads, Life Technologies or FG beads, Tamagawa seiki) at 37 ° C. for positive selection, and DMSO, SDS-containing HBS-T containing pClBz-containing HBS-T. Washed 5 times at 37 ° C. The selected cDNA on the beads is quantified by quantitative PCR (StepOnePlus, Applied Biosystems) using SYBR using T7SD8M2-Sgcc2.F53 (SEQ ID NO: 19) and G5S-4an21.R41 (SEQ ID NO: 11) as primers. Amplified with PCR Taq DNA polymerase for the next round of selection.

In the present invention, a system can be identified quickly (poly) peptide / protein tag for labeling the specifically proteins from highly diverse large libraries (D irected I n V itro E volution of R eactive peptide-tags via S equential E nrichment, to provide a DIVERS system) for short.
In this system, the displayed (poly) peptide / protein (phenotype) and its encoding DNA (genotype) are linked via a covalent bond. Only (poly) peptide / protein tags can be selected. In addition, the transcription, puromycin modification, and translation processes proceed continuously in one solution, and the reverse transcription process can be performed in a single pot without purification, so it can be performed on a large scale (about approx. ( ¹⁵ )) (poly) peptide / protein tags can be identified from the library.

Further, since the (poly) peptide / protein tag obtained by the present invention is small in size, it can be labeled with any artificial molecule without inhibiting the function of the target protein fused with the (poly) peptide / protein tag. The present invention can also be applied to a plurality of different proteins and a plurality of different labeling substances (fluorescent dyes and the like).
Furthermore, the (poly) peptide / protein tag according to the present invention can also be applied to control the activity and interaction of intracellular proteins.

Claims (11)

The following steps:
(a) a step of transcribing a DNA library to prepare an RNA library;
(b) a step of linking a nucleic acid derivative via a linker to the 3 ′ end of RNA in an RNA library to produce an RNA-linker-nucleic acid derivative library;
(c) RNA-linker-nucleic acid derivative library, using the RNA portion of the library as mRNA, peptide or protein synthesis in a cell-free translation system to produce an mRNA display peptide / protein library,
(d) a step of reverse-trancribing the mRNA portion of the mRNA display peptide / protein library to DNA to produce a cDNA display peptide / protein library;
(e) contacting the immobilized target substance with a cDNA display peptide / protein library; and
(f) selecting a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library is covalently bound to the target substance;
A method for selecting a peptide or protein that can be covalently bound to a target substance. The method of claim 1, wherein step (f) comprises washing the conjugate with an organic solvent and / or a denaturing agent. The following steps:
3. The method of claim 1 or 2, further comprising (g) amplifying the cDNA of the selected conjugate.   The method according to any one of claims 1 to 3, wherein the steps (a) to (g) are repeated. The following steps:
5. The method according to claim 3 or 4, further comprising the step of (h) determining the sequence of the amplified cDNA.   6. The method according to any one of claims 1 to 5, wherein the nucleic acid derivative is puromycin.   A peptide / protein tag containing a peptide or protein that can be covalently bound to a target substance, selected by the method according to any one of claims 1 to 6.   8. The peptide / protein tag according to claim 7, wherein the peptide or protein capable of covalently binding to the target substance contains at least one cysteine residue.   A nucleic acid display peptide / protein in which a peptide or protein capable of covalently binding to a target substance selected by the method according to any one of claims 1 to 6 and a nucleic acid encoding the peptide or protein are linked.   10. A library comprising a plurality of nucleic acid display peptides / proteins according to claim 9, wherein the nucleic acid display peptides / proteins have different nucleic acid sequences. The following steps:
(a) a step of transcribing a DNA library to prepare an RNA library;
(b) a step of linking a nucleic acid derivative via a linker to the 3 ′ end of RNA in an RNA library to produce an RNA-linker-nucleic acid derivative library;
(c) RNA-linker-nucleic acid derivative library RNA is used as mRNA to synthesize peptides or proteins in a cell-free translation system to produce an mRNA display peptide / protein library,
(d) a step of reverse-trancribing the mRNA portion of the mRNA display peptide / protein library to DNA to produce a cDNA display peptide / protein library;
(e) contacting the immobilized target substance with a cDNA display peptide / protein library;
(f) selecting a conjugate in which the peptide part or protein part of the cDNA display peptide / protein library and the target substance are covalently bound, and
(g) A method for obtaining a nucleic acid encoding a peptide or protein capable of covalently binding to a target substance, which comprises amplifying cDNA of a selected conjugate.