JP6202707B2 - Novel cell membrane penetrating peptide - Google Patents

Description

  The present invention relates to a cell membrane-penetrating peptide that is rich in histidine residues. Furthermore, the present invention also relates to mass transport through cell membranes using such peptides.

  Peptides rich in basic amino acids arginine and lysine exhibit relatively good cell membrane permeation under physiological conditions and are considered to be used as drug transport carriers (Non-patent Documents 1 to 7). .

  However, in order to realize efficient drug transport and the like, it is necessary to search for a peptide exhibiting better cell membrane permeation.

Dennison, S.R. et al. Biochem. And Biophy. Res. Comm. 363, 178 (2007) Futaki, S. Advanced Drug Delivery Rev. 57, 547 (2005) Kameyama, S. et al. Mol. Pharm. 3, 174 (2006) Futaki, S. et al. J. Biol. Chem. 276, 5836 (2001) Futaki, S. et al. J. Molec. Recognition 16, 260 (2003) Moosic, J.P. et al. Vaccine 129, 517 (1983) Nakase, I. et al. Advanced Drug Delivery Reviews 60, 598 (2008)

  It was an object of the present invention to obtain a peptide exhibiting better cell membrane penetration than conventional ones.

  The present inventors have intensively studied to solve the above-mentioned problems, and found that a peptide rich in histidine residues shows very good membrane permeation, thereby completing the present invention. Accordingly, the present invention provides the following.

(1) A transmembrane peptide having a length of 12 amino acids or more and 80% or more of amino acid residues are histidine residues.
(2) The peptide according to (1), wherein 90% or more of the amino acid residues are histidine residues.
(3) The peptide according to (1), wherein all amino acid residues are histidine residues.
(4) The peptide according to (3), which is 12 to several tens of amino acids in length.
(5) The peptide according to (4), which is 14 to several tens of amino acids in length.
(6) A construct comprising the peptide according to any one of (1) to (5) and a substance to be transported into a cell.
(7) The construct according to (6), wherein the peptide is as described in (4) or (5).
(8) The construct according to (6) or (7), wherein the substance to be transported is a drug.
(9) The construct according to (6) or (7), wherein the substance to be transported is a protein.
(10) A method for transporting a substance into a cell, comprising the following steps:
A substance to be transported into the cell is bound to the peptide according to any one of (1) to (5) to obtain a construct,
The resulting construct is introduced into cells.
(11) The method according to (10), wherein the peptide is as described in (4) or (5).
(12) The method according to (10) or (11), wherein the substance to be transported is a drug.
(13) The method according to (10) or (11), wherein the substance to be transported is a protein.
(14) The peptide according to any one of (1) to (5), the construct according to any one of (6) to (9), or (10) to (10), wherein the peptide forms a complex with a metal. The method according to any one of 12).
(15) The construct according to any one of (6) to (9) or the method according to any one of (10) to (12), wherein the peptide is as described in (4) or (5).
(16) A method for producing protein extracellularly, the method comprising the following steps:
A polynucleotide encoding the peptide according to any one of (1) to (5) and a polynucleotide encoding a protein are combined, incorporated into a vector, introduced into a cell,
The fusion protein is expressed in the cell, and the fusion protein is released outside the cell membrane.
(17) The method according to (16), wherein the peptide is as described in (4) or (5).

  Since the peptide of the present invention exhibits very efficient membrane permeation, efficient drug transport can be realized by binding a drug to the peptide of the present invention. In addition, if a complex is formed between the histidine residue of the peptide of the present invention and a metal, the drug can be transported to cells at a specific pH depending on the type of metal. Furthermore, the peptide of the present invention and a substance to be purified can be bound and applied to a metal chelate column to efficiently purify various substances.

FIG. 1 is a graph showing the cell membrane permeability of the peptide of the present invention compared to octaarginine (uptake ratio is 1). The left panel shows the case where the human macrophage-derived cell line RAW264 is used, and the right panel shows the case where the human liver cancer-derived cell line HepG2 is used. FIG. 2 is a graph comparing the cell membrane permeability of the peptides of the present invention with the cell membrane permeability of known cell membrane permeable peptides. Data from known cell-membrane-penetrating peptides are quoted from Shiro Futaki (Kyoto University), a research project "Sorst" Research Completion Report, "Peptide for Targeting Cells: Functional Analysis and Control" . FIG. 3 is a graph showing the peptide of the present invention consisting of histidine residues compared to octaarginine (incorporation rate is 1). As a cell, a human liver cancer-derived cell line HepG2 was used. FIG. 4 is a graph showing the cell membrane permeability of the peptide of the present invention compared to octaarginine (uptake ratio is 1). FIG. 5 is a photograph showing cell membrane permeation of a fusion protein (molecular weight: about 70 kDa) obtained by adding the peptide of the present invention (sequence 9) to green fluorescent protein.

  The present invention provides a peptide having a length of several amino acids or more, preferably 12 amino acids or more, and having more than half of the constituent amino acids being histidine. Such a peptide of the present invention has excellent cell membrane permeability.

  The constituent amino acids of the peptide of the present invention are rich in histidine, preferably about 80% or more of the constituent amino acid residues are histidine residues, more preferably about 90% or more of the constituent amino acid residues are histidine residues. More preferably, all constituent amino acid residues are histidine residues.

  The length of the peptide of the present invention is 12 amino acids or more, preferably 14 amino acids or more, more preferably 16 amino acids or more. Therefore, the length of the peptide of the present invention may be, for example, in the range of 12 amino acids to several tens of amino acids, preferably 14 amino acids to several tens of amino acids, more preferably 16 amino acids to several tens of amino acids. Here, several tens of amino acids mean any number of amino acids in the range of 13 to less than 100.

  As a particularly preferred example of the peptide of the present invention, all the constituent amino acid residues are histidine residues, and the length is 12 amino acids to several tens of amino acids, preferably 14 amino acids to several tens of amino acids. -30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, etc., and may be, for example, 14 amino acids to 30 amino acids, 40 amino acids, 50 amino acids, 60 amino acids, 70 amino acids, etc. .

  The amino acid residue other than histidine constituting the peptide of the present invention may be any amino acid residue, natural amino acid residue, non-natural amino acid residue, modified amino acid residue, or synthetic amino acid residue. Good. A person skilled in the art can appropriately synthesize and modify amino acids. Preferably, the amino acid residues other than histidine constituting the peptide of the present invention are basic amino acid residues such as arginine and lysine, or amino acid residues having properties similar to histidine.

  The peptide of the present invention can be prepared by a known peptide synthesis method such as the Fmoc solid phase synthesis method. Alternatively, the peptide of the present invention can also be prepared by a genetic recombination method.

  As described above, the peptide of the present invention has a high cell membrane permeability and exhibits a cell membrane permeability of about 2 to 40 times or more compared with peptides that have been conventionally known to have a high membrane permeability (FIG. 2). reference). Therefore, if the peptide of this invention is used, in the mass transport through a cell membrane, a much more efficient mass transport can be performed compared with the conventional same kind of method.

  The substance transport to cells using the peptide of the present invention will be described. First, a construct is obtained by binding the peptide of the present invention and a substance to be transported. Therefore, the substance to be transported may be bound to any residue of the peptide of the present invention, but is generally bound to the N-terminus or C-terminus of the peptide of the present invention. The peptide of the present invention and the substance to be transported may be directly bound, or may be bound using a spacer or the like. The bond may be a covalent bond, an ionic bond, a hydrophobic bond, an adsorption, or the like, and is not particularly limited. In addition, it is possible to adopt a binding mode in which the peptide of the present invention is released after being introduced into a cell. Such bonding can be appropriately performed by those skilled in the art.

  The substance to be transported is not particularly limited, and may be, for example, a physiologically active substance, a drug, an enzyme or other protein.

  Next, the obtained construct is introduced into a target cell. The peptide of the present invention can be applied to a wide variety of cells without limitation as long as it is an animal cell, but an adherent cell line is preferred. Since the peptide of the present invention shows high permeability to the cell membrane of various cancer cells, it is also useful for drug delivery to cancer cells. Conditions for introducing the construct into the cell are also known to those skilled in the art or can be easily determined by those skilled in the art. The temperature and pH at the time of introducing the construct into the cell can also be set in a wide range. For example, the construct of the present invention may be introduced into cells using the peptide of the present invention under conditions near neutrality at room temperature, or the construct may be introduced into cells in culture.

  Furthermore, the target substance can be produced extracellularly by using the peptide of the present invention in a genetic recombination method. A polynucleotide encoding the peptide of the present invention and a polynucleotide encoding a target substance such as a protein or peptide are combined and incorporated into a vector, introduced into a cell, a fusion protein is expressed in the cell, and the cell membrane is passed through. The fusion protein can be released out of the cell.

  Furthermore, since histidine forms a complex between its imidazole group and a metal such as copper, zinc, nickel, and cobalt, the peptide of the present invention also forms a metal complex. Under acidic conditions, the coordination bond between histidine and the metal ion is dissociated, and the peptide of the present invention is in a positively charged form, so that it exhibits membrane permeability. Moreover, the pH at which the coordination bond dissociation between histidine and the metal ion varies depending on the type of metal. Therefore, if a complex is formed between the peptide of the present invention and a metal, the substance can be permeated through a membrane at a desired pH condition.

  For example, in tumor tissue, tumor cells proliferate randomly, resulting in hypoxic conditions, lactic acid fermentation, and a low pH environment. Thus, using a complex of a metal of the present invention that undergoes dissociation of coordination bonds under low pH conditions and drug delivery specific to tumor tissue becomes possible.

  A further use of the peptides of the present invention is in the purification of proteins using metal chelate columns. The fusion protein of the peptide of the present invention and the target protein can be applied to a metal chelate column and adsorbed, and the target protein can be eluted under appropriate elution conditions.

  Hereinafter, the present invention will be described specifically and in detail with reference to examples. However, the examples are merely illustrative and do not limit the present invention.

Arg-His hybrid peptides containing different numbers of arginine residues and histidine residues (sequences 1 to 15; SEQ ID NOs: 1 to 15 in the sequence listing) are chemically synthesized by Fmoc solid phase synthesis, and the C-terminus is amidated. Modification, N-terminus was labeled with tetramethylrhodamine (TAMRA) fluorescent dye. The sequence of the synthesized peptide is shown below.

Under two conditions of pH 6.0 and pH 7.4, the cellular uptake of the 15 peptide sequences was quantified with a flow cytometer. The cell lines were human macrophage-derived cells: RAW264 (RIKEN cell bank: RCB0535) and human liver cancer cells: HepG2 (RIKEN cell bank: RBC1886). Both cell lines were seeded at a cell density of 5.0 × 10 ⁵ cells / well (9.6 cm ² ) in a 6-well multiplate, and a cell culture medium (pH 6.0 or pH 7.4) containing 10 μM of each peptide. Incubated for 24 hours under conditions of 37 ° C. and 5% CO ₂ concentration. Thereafter, the cell culture medium was removed, the cells were washed with 10 mM phosphate buffer, and the cells were detached from the plate with 0.05% trypsin (containing 5.3 mM EDTA). The cells were collected in a centrifuge at 500 g × 5 minutes and suspended in 500 uL of FACS buffer (flow cytometry buffer = 10 mM phosphate buffer containing 2% fetal calf serum) Cell membrane permeation efficiency of each peptide sequence was evaluated by measuring the intensity of intracellular fluorescence (TAMRA-derived red fluorescence) by flow cytometry.

  The experimental results are shown in FIG. For all peptide sequences used in the above experiments, cell membrane permeation efficiency was compared under both conditions of pH 6.0 and pH 7.4. Compared to sequence 1 (known octaarginine), cell membranes with significantly higher sequence 9 (peptide consisting of 16 histidine residues) in both pH 6.0 and pH 7.4 and in both RAW264 and HepG2 cell lines It became clear that transmission efficiency was shown. The relative cell membrane permeation efficiency of sequence 9 relative to sequence 1 is 3.13 times (pH 7.4) and 2.35 times (pH 6.0) for RAW264 and 5.95 times (pH 7) for HepG2. 4) and 5.55 times (pH 6.0) (FIG. 1).

Results of measurement of cell membrane permeation efficiency of peptide of sequence 9 and peptide of sequence 1 at pH 6.0 and 7.4 using HepG2 cells and data on cell membrane permeation efficiency of known cell membrane permeation peptides (Chinese hamster ovary cells : Measured at pH 7.4 using CHO-K1 cells) is shown in FIG. In addition, the known cell membrane permeation peptide used for the comparison is as follows.

  The peptide of the present invention (sequence 9, peptide consisting of 16 histidine residues) showed remarkable cell membrane permeation efficiency as compared with sequence 1 and known cell membrane permeation peptides. The peptide of sequence 9 was found to have an intracellular introduction efficiency about 40 times that of HIV Tat (48-60) and about 2 times that of FHV coat (35-49).

Since the peptide consisting of 16 histidine residues showed the best membrane permeability, the membrane permeability of peptides consisting of histidine residues of various lengths was examined. Peptides containing different numbers of histidine residues (sequences 1, 9, 16-24, SEQ ID NOs: 1, 9, 16-24 in the sequence listing) were chemically synthesized by Fmoc solid phase synthesis, and the C-terminal was amidated Modification, N-terminus was labeled with tetramethylrhodamine (TAMRA) fluorescent dye. The sequence of the synthesized peptide is shown below.

  The experiment was performed in the same manner as in Example 1 using HepG2 cells. The results are shown in FIG. Under both conditions of pH 6.0 and 7.4, when the number of histidine residues was 12 or more, membrane permeability was remarkably increased. The permeability of the peptide having 12 histidine residues (sequence 19) was about 2.8 times (pH 7.4) and about 1.2 times (pH 6.0) that of octaarginine (sequence 1). The membrane permeability of the peptide having 14 histidine residues (sequence 20) was about 3.2 times (pH 7.4) and about 3.4 times (pH 6.0) that of octaarginine (sequence 1). The membrane permeability of the peptide having 16 histidine residues (sequence 9) was about 5.3 times (pH 7.4) and about 4.7 times (pH 6.0) that of octaarginine (sequence 1). The permeability of the peptide having 18 to 24 histidine residues (sequence 21 to 24) is almost the same as that of the peptide having 16 histidine residues (sequence 9), and octaarginine (sequence 1) at any pH. About 4.5 times to about 4.9 times.

Under the condition of pH 7.4, the amount of intracellular uptake of 11 kinds of peptide sequences of sequences 1, 9, and 16 to 24 was quantified with a flow cytometer. The cell lines used were human Burkitt lymphoma cells: RAJI (RIKEN cell bank: RCB1647), human T-lymphocytic leukemia cells: Jurkat (RIKEN cell bank: RCB0806), human glioma cells: U251 (RIKEN cell bank: RCB0461). ), Mouse macrophage cells: RAW264 (RIKEN cell bank: RCB0535), human liver cancer cells: HepG2 (RIKEN cell bank: RCB1886), mouse fibroblasts: NIH-3T3 (RIKEN cell bank: RCB2767), human fibrosarcoma cells: HT1080 (RIKEN cell bank: RCB1956) and human squamous cell carcinoma cells: RERF (RIKEN cell bank: RCB0444). Each cell line was seeded in a 6-well multiplate so that the cell density was 5.0 × 10 ⁵ cells / well (9.6 cm ² ), and cell culture medium containing 10 μM of each peptide (adjusted to pH 7.4). Incubated for 3 hours at 37 ° C. and 5% CO ₂ concentration. Thereafter, the cell culture medium was removed, the cells were washed with 10 mM phosphate buffer, and the cells were detached from the plate with 0.05% trypsin (containing 5.3 mM EDTA). The cells were collected in a centrifuge at 500 g × 5 minutes and suspended in 500 μL of FACS buffer (flow cytometry buffer = 10 mM phosphate buffer containing 2% fetal calf serum) Cell membrane permeation efficiency of each peptide sequence was evaluated by measuring the intensity of intracellular fluorescence (TAMRA-derived red fluorescence) by flow cytometry.

  The results are shown in FIG. The peptide of the present invention is generally difficult to permeate the cell membrane for suspension cell lines (RAJI or Jurkat), and conversely to adhesion cell lines (U251, RAW264, HepG2, NIH-3T3, HT1080, RERF). On the other hand, it became clear that it tends to permeate the cell membrane. In particular, when compared with octaarginine (sequence 1), which is an existing cell membrane-penetrating peptide, the peptide of the present invention has a marked increase in cell membrane permeability to adherent cell lines when the number of histidine residues is 12 or more. did. The maximum permeability of the peptide having 12 histidine residues (sequence 19) was about 5.2 times that of octaarginine (sequence 1). The membrane permeability of the peptide having 14 histidine residues (sequence 20) was about 13.2 times that of octaarginine (sequence 1) at maximum. The membrane permeability of the peptide having 16 histidine residues (sequence 9) was about 17.1 times that of octaarginine (sequence 1) at maximum. The peptide having 18 histidine residues (sequence 21) had a maximum membrane permeability of about 9.7 times that of octaarginine (sequence 1). The maximum permeability of the peptide having 20 histidine residues (sequence 22) was about 18.6 times that of octaarginine (sequence 1). The membrane permeability of the peptide having 22 histidine residues (sequence 23) was about 27.6 times that of octaarginine (sequence 1) at maximum. The membrane permeability of the peptide having 24 histidine residues (sequence 24) was about 23.8 times that of octaarginine (sequence 1) at maximum.

To add a peptide having 16 histidine residues (sequence 9) to the C-terminal part of GFP, which is a green fluorescent protein, and to facilitate further purification, maltose-binding protein (MBP) is added to the N-terminal part of GFP. With the added, the recombinant protein was expressed in E. coli. The expression plasmid used at this time was pMal-c2x, and the Escherichia coli strain was BL21. The fusion protein obtained by expression: MBP-GFP-H16 (molecular weight: about 70 kDa) was purified by affinity purification using the affinity between MBP and amylose resin. Human fibrosarcoma cells: HT1080 (RIKEN Cell Bank: RCB1956) seeded at a cell density of 5.0 × 10 ⁴ cells / well (0.32 cm ² ) in a multiwell glass bottom dish, and a fusion protein obtained by purification Incubation was carried out in cell culture medium (adjusted to pH 7.4) containing 5 μM for 3 hours at 37 ° C. and 5% CO ₂ concentration. Thereafter, the cell culture medium was removed, the cells were washed with 10 mM phosphate buffer, new cell culture medium was added again, and intracellular green fluorescence was observed and photographed under a confocal laser microscope.

  The results are shown in FIG. Only very weak autofluorescence was observed from untreated cells. On the other hand, clear intracellular fluorescence was observed from cells treated with the fusion protein added with the peptide of the present invention (sequence 9): MBP-GFP-H16 (molecular weight: about 70 kDa). From this, it was shown that the target protein can be delivered into cells by using the peptide of the present invention.

  Since the peptides of the present invention exhibit very efficient membrane permeation, for example, efficient drug transport can be realized. In addition, if a complex is formed between the histidine residue of the peptide of the present invention and a metal, the drug can be transported to cells at a specific pH depending on the type of metal. Furthermore, various substances can be efficiently purified by applying the peptide of the present invention to a metal chelate column. Therefore, the present invention provides a peptide that enables a substance to permeate the cell membrane very efficiently, and can be used in the fields of medicine, research, protein production, and the like.

Claims (11)

Is not less 12 or more amino acids in length, all of the amino acid residues includes a Ru film penetrating peptides der histidine residues, and a protein to be transported into the cell, constructs with cell membrane permeability.   The construct according to claim 1, wherein the length of the peptide is 12 to several tens of amino acids.   The construct according to claim 2, wherein the peptide has a length of 14 to several tens of amino acids.   The construct according to any one of claims 1 to 3, wherein the peptide forms a complex with a metal. A method for transporting proteins into cells, comprising the following steps (but not performed in the human body) :
And protein to be transported into the cell, is not less 12 or more amino acids in length, to give a construct all of the amino acid residues are bound histidine residues der Ru transmembrane peptide, then
The resulting construct is introduced into cells.   The method according to claim 5, wherein the peptide has a length of 12 to several tens of amino acids.   The method according to claim 6, wherein the peptide has a length of 14 to several tens of amino acids.   The method according to any one of claims 5 to 7, wherein the peptide forms a complex with a metal. A method for producing protein extracellularly, comprising the following steps:
Is not less 12 or more amino acids in length, and introduced into cells of all the polynucleotides which amino acid residues encoding Der Ru transmembrane peptide histidine residues incorporated into a vector by coupling the polynucleotide encoding a protein Then
The fusion protein is expressed in the cell, and the fusion protein is released outside the cell membrane.   The method according to claim 9, wherein the peptide has a length of 12 to several tens of amino acids.   The method according to claim 10, wherein the peptide has a length of 14 to several tens of amino acids.