Methods for introducing proteins into animal cells

Abstract

Provided is a method for introducing a protein into an animal cell, the method comprising a step for bringing the protein into contact with the animal cell in a medium having a calcium ion concentration of 0-0.3 mM. In addition, provided are: a medium for introducing a protein into an animal cell, the medium having a calcium ion concentration of 0-0.3 mM and a magnesium ion concentration of 0-2 mM; and a composition for promoting the introduction of a protein into an animal cell, the composition comprising a calcium chelating agent.

Description

[Technical field]

[0001] The present invention relates to a method for efficiently introducing a protein into an animal cell by using a medium having a reduced calcium concentration, and also to a medium used in said method. [Background technology]

[0002] Methods for introducing proteins into cells are used for genome modification, genome editing, protein function analysis, etc. Living cells have a cell membrane, and it is possible to introduce low molecular weight compounds from outside the cell, but it is known that it is difficult to introduce proteins, which are larger biomolecules, from outside the cell.

[0003] Methods for introducing proteins into cells are generally broadly divided into physical and biochemical methods. Known physical methods include electroporation and nanoneedle insertion, which create holes in the cell membrane to introduce a substance. Meanwhile, known biochemical methods include a method in which a protein is complexed with a cell introduction reagent (liposome, viral envelope, or cell membrane-permeable peptide (also called a protein transduction domain)) and then introduced into the cell, and a protein transduction method in which only the protein is introduced into the cell.

[0004] Approaches to improve the efficiency of protein introduction in protein transduction include a method of fusing a cell membrane-permeable peptide with a target protein to form a fusion protein, and a method of using a compound that improves the uptake of substances into cells. As cell membrane-permeable peptides, Tat peptide and R9 peptide are known, both of which have sequences rich in basic amino acids (Non-Patent Document 1). These sequences are expected to improve endocytosis. Meanwhile, chloroquine and pyrene butyric acid have been reported as compounds that improve the uptake of substances into cells (Non-Patent Document 2).

[0005] However, many of the conventional methods have a negative effect on cells when proteins are introduced into animal cells. For example, physicochemical methods may damage cell membranes, and methods in which the target protein is used by forming a complex with a cell introduction reagent may cause cytotoxicity. In methods using compounds that improve the uptake of substances into cells, the presence of unknown toxicity cannot be denied at this stage, because the cell damage, long-term cytotoxicity, carcinogenicity, and teratogenicity of the compounds have not been fully verified. In particular, when introducing proteins into cells for the purpose of human treatment, it is necessary to use drugs with guaranteed safety. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Naoki Kajiwara, Futoshi Shibasaki “Cell membrane permeable peptides” Japanese Pharmacological Journal 2013, 141(4), 220-221 [Non-Patent Document 2] Takeuchi et al., ACS Chem. Biol. 2006, 1(5), 299-303 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in consideration of these circumstances, and an object of the present invention is to provide a method for improving the efficiency of protein introduction into animal cells while suppressing negative effects on the cells. [Means for solving the problem]

[0008] As a result of extensive research aimed at solving the above problems, the inventors discovered that by using a medium with a reduced calcium ion concentration when introducing a protein into animal cells, it is possible to significantly improve the efficiency of protein introduction while suppressing negative effects on the cells, and thus completed the present invention.

[0009] That is, according to one embodiment, the present invention relates to a method for introducing a protein into an animal cell, the method comprising the step of contacting the protein with the animal cell in a medium having a calcium ion concentration of 0 to 0.3 mM.

[0010] The magnesium ion concentration in the medium is preferably 0 to 2 mM.

[0011] Moreover, according to one embodiment, the present invention relates to a medium for introducing a protein into an animal cell, the medium having a calcium ion concentration of 0 to 0.3 mM and a magnesium ion concentration of 0 to 2 mM.

[0012] The calcium ion concentration is preferably 0.1 to 0.2 mM.

[0013] The medium preferably contains a calcium chelating agent, such as, for example, a calcium chelating agent preferably selected from the group consisting of EDTA, EGTA and BAPTA.

[0014] According to one embodiment, the present invention also relates to a composition for promoting the introduction of a protein into an animal cell, comprising a calcium chelating agent, and such a calcium chelating agent is preferably selected from the group consisting of, for example, EDTA, EGTA and BAPTA. Effect of the Invention

[0015] The method of the present invention makes it possible to dramatically improve the efficiency of protein introduction into animal cells while suppressing negative effects on the activity and characteristics of the cells. In the present invention, the efficiency of protein introduction into animal cells can be improved simply by using a medium with a reduced calcium concentration, and no special equipment or reagents are required. Therefore, the method of the present invention is also excellent in terms of simplicity. Furthermore, it is versatile in that it does not matter what type of protein is introduced. [Brief description of the drawings]

[0016] [Figure 1A] 1B to 1D show an outline of the experimental technique used. We used reporter cells in which the DNA between the loxP sequences is removed when the Cre protein is introduced into the cells, resulting in the expression of the downstream GFP gene. [Figure 1B] This is a photograph of reporter cells showing the results of analyzing the effect of divalent cations in medium components on the introduction of Cre protein into the cell nucleus using the experimental method in Figure 1A. Verification was performed using a buffer solution (HBS), medium (DMEM), and medium (DMEM) from which divalent cations had been removed using chelating agents (EDTA, EGTA). In the figure, "Ctrl" indicates reporter cells into which Cre protein had not been introduced. [Figure 1C] This graph shows the recombination rate by Cre protein, which is the result of analyzing the effect of divalent cations in medium components on the introduction of Cre protein into the cell nucleus using the experimental method in Figure 1A. Verification was performed using a buffer solution (HBS), medium (DMEM), and medium (DMEM) from which divalent cations were removed using chelating agents (EDTA, EGTA). In the figure, "Ctrl" indicates the recombination rate in cells without Cre protein introduction. [Figure 1D] This graph shows the recombination rate by Cre protein, which is the result of analyzing the effect of divalent cations in medium components on the introduction of Cre protein into the cell nucleus using the experimental method in Figure 1A. The experiment was performed in a medium (DMEM) from which calcium ions and / or magnesium ions were removed, as well as in a medium (DMEM) from which these ions were not removed. The Cre proteins used were a fusion protein with a His tag and a fusion protein with a His tag and a nuclear localization signal (NLS) or a modified version thereof. In the figure, "Ctrl" indicates the recombination rate in cells into which Cre protein was not introduced. [Diagram 2]This graph shows the recombination rate by Cre protein, which is the result of analyzing the effect of calcium ions in the medium on the introduction of Cre protein into the cell nucleus using the experimental method in Figure 1A. The experiment was performed in a medium with and without calcium ions (DMEM). As Cre proteins, fusion proteins with His tags and fusion proteins with His tags and nuclear localization signals (NLS) and / or Tat peptides added were used at various concentrations. [Figure 3A] This is a diagram showing an outline of the experimental method used in Figures 3B and 3C. A reporter cell was used in which the HiBiT protein (the other of the split luciferases) fused with the Cre protein was introduced into cells expressing the LgBiT protein (one of the split luciferases), and when the two proteins associate and the luciferase function is restored, the reporter cell emits light in the presence of furimazine. In this reporter cell, the cytosol normally emits light, but if a nuclear localization signal is fused to the LgBiT protein, the nucleus emits light. [Figure 3B] This is a graph showing the effect of calcium ion concentration in the medium on the introduction of the Cre protein into the cytosol or nucleus of cells over time, as measured by luciferase activity, using the experimental method in Figure 3 A. Luciferase activity in the whole cell was measured using LgBiT protein not fused to a nuclear localization signal. [Figure 3C] This is a graph showing the effect of calcium ion concentration in the medium on the introduction of the Cre protein into the cytosol or nucleus of cells over time, using luciferase activity as an indicator, using the experimental method of Figure 3 A. Luciferase activity in the nucleus was measured using LgBiT protein fused with a nuclear localization signal. [Figure 3D] FIG. 1B is a photograph of reporter cells showing the results of an analysis of the effect of divalent cations in medium components on the introduction of HiBiT-fused Cre protein into the cell nucleus, using the experimental method of FIG. 1A. [Figure 3E]This graph shows the recombination rate by Cre protein, which is the result of analyzing the effect of divalent cations in the medium components on the introduction of HiBiT-fused Cre protein into the cell nucleus, using the experimental method in Figure 1 A. In the figure, "Ctrl" indicates the recombination rate in cells not introduced with Cre protein. [Figure 3F] This figure shows the structure of the Cre protein used in the experiments of Figures 3G to 3L. The Cre protein is fused to a His tag, a nuclear localization signal (NLS), a Tat peptide, and / or a HiBiT protein at various positions and in various orders. [Figure 3G] FIG. 3C is a graph showing the results of measuring luciferase activity over time in the whole cells in the presence of calcium ions by the experimental method of FIG. 3A using each Cre protein of FIG. 3F. [Figure 3H] FIG. 3C is a graph showing the results of measuring luciferase activity over time in the whole cells in the absence of calcium ions by the experimental method of FIG. 3A using each Cre protein of FIG. 3F. [Figure 3I] FIG. 3C is a graph showing the results of measuring luciferase activity in the nucleus over time in the presence of calcium ions by the experimental method of FIG. 3A using each Cre protein of FIG. 3F. [Figure 3J] FIG. 3C is a graph showing the results of measuring luciferase activity in the nucleus over time in the absence of calcium ions by the experimental method of FIG. 3A using each Cre protein of FIG. 3F. [Figure 3K] This is a photograph of reporter cells in which HiBiT-fused Cre protein was introduced into the nucleus of the cells in the presence of calcium ions, according to the experimental method of Figure 1A. In the figure, "Ctrl" indicates reporter cells in which Cre protein was not introduced. [Figure 3L] This is a photograph of reporter cells in which HiBiT-fused Cre protein was introduced into the nucleus of the cells in the absence of calcium ions, according to the experimental method of Figure 1A. In the figure, "Ctrl" indicates reporter cells in which Cre protein was not introduced. [Figure 4A]This is a graph showing the introduction of ZFN protein into the cell nucleus as a specific frameshift rate by ZFN. When ZFN protein is introduced into the cell, the ZFN recognition sequence set in the GFP gene is cut, and reporter cells were used in which the GFP gene is expressed when a specific frameshift occurs due to subsequent repair. Tests were performed in medium containing calcium ions (DMEM), medium without calcium ions (DMEM), and medium (DMEM) from which divalent cations have been removed using a chelating agent (EDTA). ZFN proteins were used at various concentrations. [Figure 4B] This is a graph showing the introduction of Cas9-guide RNA complexes into the nucleus of cells at a specific frameshift rate by CRISPR-Cas9. When the Cas9-guide RNA complex is introduced into the cell, the CRISPR-Cas9 recognition sequence in the GFP gene is cut, and a reporter cell was used in which the GFP gene is expressed when a specific frameshift occurs due to subsequent repair. The experiment was performed in a medium containing calcium ions (DMEM) and a medium not containing calcium ions (DMEM). As a control, the introduction of the Cas9-guide RNA complex into the cell was performed by lipofection. The Cas9 protein was used at various concentrations. In the figure, "-" indicates the introduction of only the Cas9 protein, "N" indicates the introduction of a complex of a guide RNA and Cas9 protein different from the target sequence, and "target" and "T" indicate the introduction of a complex of a target guide RNA and Cas9 protein, respectively. [Figure 4C]This graph shows the rate of change in luciferase activity due to transcriptional activation after Gal4-VP16 protein is introduced into the cell nucleus. Reporter cells were used in which the Gal4-VP16 protein is introduced into the cell and binds to the promoter region, causing expression of the downstream luciferase gene. Tests were performed in medium containing calcium ions (DMEM, +Ca2+) and medium not containing calcium ions (DMEM, -Ca2+). Gal4-VP16 protein was used at various concentrations. In the figure, "Ctrl" shows the results of the same procedure using medium containing calcium ions (DMEM, +Ca2+) without the addition of Gal4-VP16 protein, and "DNA" shows the results of introducing a plasmid expressing Gal4-VP16 protein by lipofection instead of Gal4-VP16 protein. [Figure 5A] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into the HEK293_F*GxFL cell line under different calcium ion concentration conditions. [Figure 5B] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into a HeLa_F*GxFL cell line under different calcium ion concentration conditions. [Figure 5C] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into the HT1080_F*GxFL cell line under different calcium ion concentration conditions. [Figure 5D] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into the MCF7_F*GxFL cell line under different calcium ion concentration conditions. [Figure 5E] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into Jurkat_F*GxFL cell line under different calcium ion concentration conditions. [Figure 5F] 1 is a graph showing the luciferase activity (relative value) of HNCre introduced into the K562_F*GxFL cell line under different calcium ion concentration conditions. [Figure 6A]1 is a graph showing cell activity after 1 hour of culture in buffer or medium without FBS. [Figure 6B] 1 is a graph showing cell activity after culturing in a medium containing FBS for 1 hour. [Figure 7] 1 is a graph showing the ratio of GFP-positive cells after introduction of NCre and Cre with or without a His tag into 293.RxG cells in the presence or absence of calcium ions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention will be described in detail below, but the present invention is not limited to the embodiments described in the present specification.

[0018] <Methods for introducing proteins into animal cells> According to a first embodiment, the present invention is a method for introducing a protein into an animal cell, the method comprising the step of contacting the protein with the animal cell in a medium having a calcium ion concentration of 0 to 0.3 mM.

[0019] There is no particular limitation on the "animal cells" used in the method of this embodiment. Examples of animal cells include cells of mammals, fish, birds, reptiles, amphibians, and insects. Here, "mammals" is a concept that includes humans and non-human mammals. Examples of non-human mammals include even-toed animals such as cows, wild boars, pigs, sheep, and goats, odd-toed animals such as horses, rodents such as mice, rats, guinea pigs, hamsters, and squirrels, lagomorphs such as rabbits, and carnivores such as dogs, cats, and ferrets.

[0020] The animal cells in this embodiment may be primary cultured cells or cell lines derived from any organ or tissue of an animal. The type of cells is not particularly limited, and may be adherent cells or suspension cells. Specific examples of animal cells include somatic cells such as fibroblasts, hematopoietic cells, T cells, B cells, neurons, cardiac muscle cells, skeletal muscle cells, bone cells, hepatic cells, pancreatic cells, brain cells, and kidney cells; stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; germ cells such as oocytes and sperm; and germ cells of embryos at various stages (for example, 1-cell stage embryos, 2-cell stage embryos, 4-cell stage embryos, 8-cell stage embryos, 16-cell stage embryos, morula stage embryos, etc.). However, application to human germ cells and embryonic cells is excluded if it is not permitted from an ethical point of view.

[0021] In the method of this embodiment, the "protein" to be introduced into the animal cell is not particularly limited. Any protein to be introduced into the animal cell can be used. Here, "protein" means a molecule in which two or more amino acids are bound by peptide bonds. Therefore, the protein in this embodiment also includes oligopeptides and polypeptides. The amino acids in the protein may be modified, and may contain unnatural amino acids. The protein may be naturally derived or artificially synthesized.

[0022] From the viewpoint of efficiency of introduction into cells, the protein in this embodiment is preferably 300 kDa or less, more preferably 250 kDa or less, and preferably has an isoelectric point (pI) of 5.0 or more, more preferably 6.0 or more, and most preferably 6.0 to 11.0.

[0023] The protein of this embodiment may have a tag for purification added to the N-terminus and / or C-terminus. Examples of tags that can be used for purification include His tag, GST tag, HA tag, and FLAG tag. The protein of this embodiment may be modified in various ways depending on the purpose, but it is preferable that the protein is not modified in such a way that excessive positive or negative charges are imparted to the entire protein, for example, by introducing a large number of positively or negatively charged amino acids.

[0024] The protein in this embodiment may be, but is not limited to, an enzyme such as nuclease, recombinase, integrase, deaminase, methyltransferase, methylcytosine hydrogenase, ligase, glycosylase, or a transcription factor. In the method of this embodiment, a single protein selected from the above may be used, or two or more proteins may be used in combination. In addition, the protein may be bound to another substance, or may form a complex with another substance. Examples of substances that bind to proteins include various labeling compounds, and examples of substances that form complexes with proteins include nucleic acids such as RNA and DNA, in addition to proteins.

[0025] According to certain embodiments, various genome editing systems can be used as proteins to be introduced into animal cells to modify the genome. Examples of genome editing systems include fusion proteins in which a DNA binding domain such as ZF (zinc finger), TALE (TAL effector), or PPR (pentatricopeptide repeat) is bound to a DNA modification domain such as a nuclease, and a protein and RNA complex such as CRISPR / Cas. Examples of CRISPR / Cas include, but are not limited to, class 2 / II type CRISPR / Cas such as CRISPR-Cas9, class 2 / V type CRISPR / Cas such as CRISPR-Cpf1 (Cas12a), CRISPR-Cas12b, CRISPR-CasX (Cas12e), and CRISPR-Cas14, class 2 / VI type CRISPR / Cas such as CRISPR-Cas13a, and class 1 / I type CRISPR / Cas such as CRISPR-Cas3. CRISPR / Cas may be a genome editing method that uses the activity of other proteins, not the nuclease activity of Cas, by fusing other proteins to Cas (e.g., nCas, dCas) with reduced nuclease activity. Examples of the activity of other proteins fused to Cas include, but are not limited to, nuclease activity, deaminase activity (e.g., cytidine deaminase activity, adenosine deaminase activity), methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer formation activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, photolyase activity, glycosylase activity, transcription activity, reverse transcription activity, and transcription repression activity.

[0026] For examples of genome editing systems using ZF, see International Publication No. WO 2003 / 087341. For examples of genome editing systems using TALE, see International Publication No. WO 2012 / 104729, International Publication No. WO 2011 / 072246, International Publication No. WO 2015 / 019672. For examples of genome editing systems using PPR, see Nakamura et al., (2012) Plant Cell Physiol 53:1171-1179, International Publication No. WO 2014 / 175284. For examples of genome editing systems using CRISPR / Cas, see International Publication No. WO 2014 / 093712, International Publication No. WO 2013 / 176772, International Publication No. WO 2013 / 142578, International Publication No. WO 2016 / 205711, Strecker J, et al., Nature Communications 10:212(2019); Liu JJ, et al., Nature 566:218-223(2019); International Publication No. 2018 / 225858).

[0027] When modifying a genome, a genome modification enzyme other than the above genome editing system can be used as a protein to be introduced into an animal cell. For example, by using a Cre protein as a genome modification enzyme, a sequence sandwiched between recognition sequences (loxP) on a target nucleic acid can be removed, which is useful for producing conditional knockout animals. As a genome modification enzyme, various other recombinases, resolvases, transposases, etc. can also be used in addition to the Cre protein.

[0028] The medium used in the method of the present embodiment may be any medium as long as the calcium ion concentration is less than 0.3 mM. In the present embodiment, the term "medium" refers to a solution containing nutrients that allow animal cells to grow, regardless of whether the solution is named as a medium. Therefore, for example, a solution to which necessary nutrients have been added to a buffer solution may also be included in the medium of the present embodiment. The nutrients are typically carbon sources, nitrogen sources, vitamins, and inorganic salts. The nutrients vary depending on the type of medium, and examples of carbon sources include sugars such as glucose, galactose, maltose, fructose, and inositol, fatty acids such as linoleic acid and lipoic acid, and organic substances such as ethanolamine, glutathione, hypoxanthine, putrescine, pyruvic acid, citric acid, and thymidine, and examples of nitrogen sources include glycine, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, and the like. Examples of the amino acids include amino acids such as selenium, proline, serine, threonine, tryptophan, tyrosine, valine, hydroxyproline, glutamine, and selenocysteine; examples of the vitamins include ascorbic acid, biotin, choline, pantothenic acid, folic acid, inositol, niacin, pyridoxal, riboflavin, thiamine, and vitamin B12; examples of the inorganic salts include salts containing calcium, magnesium, copper, iron, potassium, sodium, zinc, manganese, chlorine, phosphate, sulfate, carbonate, and nitrate. Examples of the culture medium for animal cells include minimum essential medium (MEM), Dulbecco's modified Eagle medium (DMEM), Opti-MEM (registered trademark), and RPMI, and the calcium concentration of these media may be reduced to less than 0.3 mM. Nutrients may also be added to calcium ion-free buffer solutions such as PBS and HBS.

[0029] The calcium ion concentration of the medium in this embodiment is 0 to 0.3 mM, preferably 0.004 to 0.2 mM, and more preferably 0.1 to 0.2 mM. By using such a calcium ion concentration, the efficiency of protein introduction into animal cells can be dramatically improved. In contrast, conventional culture media for animal cells contain higher concentrations of calcium ions, which reduces the efficiency of protein introduction into animal cells. In addition, using a simple buffer solution instead of a culture medium is not preferable because it has a negative effect on the activity (e.g., proliferation activity) of the animal cells.

[0030] The concentration of metal ions other than calcium ions in the medium in this embodiment is not particularly limited, but is preferably about the same as that in a normal medium. Therefore, the magnesium ion concentration in the medium in this embodiment is preferably 0 to 2 mM, for example, may be 0.04 to 2 mM, and is particularly preferably 0.4 to 1 mM.

[0031] The calcium concentration in the medium can also be reduced by adding a calcium chelating agent to the medium. The "calcium chelating agent" in this embodiment is not particularly limited as long as it can chelate calcium ions, and examples thereof include, but are not limited to, biodegradable chelating agents such as glycol ether diamine tetraacetic acid (EGTA), ethylene diamine tetraacetic acid (EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA), 1,6-hexamethylene diamine-N,N,N',N'-tetraacetic acid (HDTA), O-ethyl S,S-diphenyl phosphorodithioate (EDDP) and derivatives thereof, and 3-hydroxy-2,2'-iminodisuccinic acid (HIDS). In addition, the calcium chelating agent in this embodiment may be a fluorescent calcium indicator such as Fura-2, Fluo-4, Indo-1, Rhod-2, etc. In the method of this embodiment, a single calcium chelating agent selected from the above may be used, or two or more calcium chelating agents may be used in combination.

[0032] The addition of a calcium chelating agent captures calcium ions, resulting in a trace amount of free calcium ions. For example, the conditional formation constant of EDTA for calcium ions at pH 7.4 is logK'=7.8. In this case, if 1 mM EDTA is present for 1 mM calcium ions, the free calcium ion concentration can be calculated to be 0.004 mM. EDTA can also capture magnesium ions, and for example, if 1 mM EDTA is present for 1 mM magnesium ions at pH 7.4 (logK'=5.8), the free magnesium ion concentration can be calculated to be 0.04 mM.

[0033] The calcium chelating agent in this embodiment is preferably one having high selectivity for calcium ions, and specifically, is preferably selected from the group consisting of EDTA, EGTA, and BAPTA. The calcium chelating agent in this embodiment is preferably added to the medium at a concentration of, for example, 0.5 to 5 mM.

[0034] The contact between the protein and the animal cells in the medium can be carried out by mixing the protein, the animal cells, and the medium and incubating for a certain period of time. The order of mixing the three is not important, and the three may be mixed simultaneously, the protein and the medium may be mixed and then mixed with the animal cells, the animal cells and the medium may be mixed and then mixed with the protein, or the protein and the animal cells may be mixed and then mixed with the medium. The incubation time may be, for example, 1 minute to 72 hours. The incubation temperature may be appropriately set depending on the type of cells used, and may be, for example, 37°C for mammalian cells. It is preferable to wash the animal cells with the medium before contacting the protein with the animal cells in the medium.

[0035] The concentration of the protein in the medium when contacting with the animal cells is not particularly limited, but is usually 0.02 μM to 100 μM, preferably 0.1 μM to 100 μM. The protein concentration may be 100 μM or more, and generally, the higher the protein concentration, the greater the number of protein molecules introduced into the cytosol, but depending on the properties of the protein (e.g., solubility, viscosity, etc.), this may have a negative effect on the introduction efficiency. Those skilled in the art can appropriately select an appropriate protein concentration depending on the protein, cells, medium, etc. used.

[0036] According to the method of the present embodiment, a protein can be introduced into an animal cell with high efficiency. The introduction of the protein into the animal cell is a so-called protein transduction that utilizes the phenomenon that the protein is spontaneously taken up into the cell, and does not require the use of special devices or reagents. There have been several reports of protein transduction in mammalian cells, but in the present invention, it has been revealed that the efficiency of protein introduction can be dramatically improved by using a medium with reduced calcium ions. However, as long as a medium with reduced calcium ions is used, there is no restriction on combining it with other physicochemical or biochemical methods, and it is possible to improve the efficiency of protein introduction compared to the case of using these methods alone.

[0037] The method of the present embodiment may be combined with other methods for improving the efficiency of protein transduction. For example, such methods include fusing a cell membrane-permeable peptide (also called a protein transduction domain) with a target protein. As cell membrane-permeable peptides, for example, Tat peptide and R9 peptide are known, both of which have sequences rich in basic amino acids. In fact, in the examples of the present application, it has been shown that the efficiency of introduction of a protein fused with a cell membrane-permeable peptide (Tat peptide) into animal cells is significantly improved by using a medium with a reduced calcium ion concentration compared to the case of using a medium without a reduced calcium ion concentration (FIG. 2).

[0038] <Culture medium and composition for introducing proteins into animal cells> According to a second embodiment, the present invention provides a medium for introducing a protein into an animal cell, the medium having a calcium ion concentration of 0 to 0.3 mM and a magnesium ion concentration of 0 to 2 mM. In this embodiment, the terms "protein", "animal cell" and "medium" are the same as those defined in the first embodiment. The calcium ion concentration of the medium in this embodiment is preferably 0.1 to 0.2 mM.

[0039] The medium of this embodiment may contain a calcium chelating agent. The "calcium chelating agent" in this embodiment is the same as that defined in the first embodiment. In the medium of this embodiment, a single calcium chelating agent as defined in the first embodiment may be used, or two or more calcium chelating agents may be used in combination. The calcium chelating agent in this embodiment is preferably selected from the group consisting of EDTA, EGTA, and BAPTA. The calcium chelating agent in this embodiment is preferably added to the medium at a concentration of, for example, 0.5 to 5 mM.

[0040] The medium of the present embodiment is advantageous in that it can increase the efficiency of introducing proteins into animal cells while suppressing negative effects on the activity and characteristics of the animal cells.

[0041] According to a third embodiment, the present invention provides a composition for promoting the introduction of a protein into an animal cell, comprising a calcium chelating agent. In this embodiment, the terms "protein," "animal cell," and "calcium chelating agent" are the same as those defined in the first embodiment.

[0042] The composition of the present embodiment may contain one or more calcium chelating agents selected from the calcium chelating agents defined in the first embodiment, and preferably contains a calcium chelating agent selected from the group consisting of EDTA, EGTA, and BAPTA. The composition of the present embodiment may be composed of only a calcium chelating agent, but generally may further contain known physiologically acceptable carriers and additives as optional components. For example, when the composition of the present embodiment is made into a liquid preparation, sterilized water, physiological saline, etc. can be used as a carrier.

[0043] The composition of this embodiment may be added to a culture medium so that the calcium ion concentration is less than 0.3 mM. EXAMPLES

[0044] The present invention will be further described below with reference to examples, but the present invention is not limited thereto.

[0045] A. Materials and Methods (1) Protein preparation The proteins used in this example were expressed using a pET-derived expression vector. The expression vector was transformed into E. coli strain BL21 (DE3). The resulting transformant was shake-cultured in LB medium at 37°C for 3 hours, and then IPTG was added at a final concentration of 0.1 mM, followed by shaking culture at 18°C ​​overnight. The collected E. coli was dissolved in a lysis buffer (10 mM Tris-HCl, 500 mM NaCl, 10% glycerol, 10 mM imidazole, 1 mM benzylsulfonyl fluoride, 1 mM dithiothreitol, pH 8.0) and purified using a Ni-NTA column to obtain a crude His-tagged protein. When the His tag was to be removed, the His-tagged protein was cleaved with a His-tag-fused SUMO protease, and the cleavage product and protease were removed using a Ni-NTA column to obtain a crude His-tagged protein. The crude product was subjected to gel filtration chromatography using Buffer A (20 mM HEPES, 500 mM NaCl, 10% glycerol, pH 7.4) or buffer exchange by ultrafiltration, and then stored at -80°C.

[0046] The amino acid sequences of the proteins prepared in this example are shown below.

[0047] HNCre protein (41.1 kDa, pI= 9.80) (SEQ ID NO: 1) [ka]

[0048] HCre protein (40.2 kDa, pI= 9.50) (SEQ ID NO: 2) [ka]

[0049] HTCre protein (41.5 kDa, pI= 10.07) (SEQ ID NO: 3) [ka]

[0050] HTNCre protein (42.4 kDa, pI= 10.24) (SEQ ID NO: 4) [ka]

[0051] HBNCre protein (43.2 kDa, pI= 9.94) (SEQ ID NO: 5) [ka]

[0052] Cre(ΔHis) protein (39.2 kDa, pI= 9.50) (SEQ ID NO: 6) [ka]

[0053] NCre(ΔHis) protein (40.1 kDa, pI= 9.80) (SEQ ID NO: 7) [ka]

[0054] ZFN protein (39.9 kDa, pI= 9.61) (SEQ ID NO: 8) [ka]

[0055] Cas9 protein (163.3 kDa, pI= 9.20) (SEQ ID NO: 9) [ka]

[0056] Gal4-VP16 protein (24.9 kDa, pI= 6.20) (SEQ ID NO: 10) [ka]

[0057] In addition, a guide RNA (32.4 kDa, pI ∼5) (SEQ ID NO: 11) to be complexed with the Cas9 protein was prepared by in vitro transcription reaction using T7 RNA polymerase. [ka]

[0058] (2) Preparation of cells transiently expressing GeLgBiT reporter pFT-GeLgBiT was prepared by inserting a fragment of the GeLgBiT gene into the pFT vector (Furuhata et al., Sci. Rep. 2019, 9, 2163). GeLgBiT (SEQ ID NO: 12) was prepared by fusing the mNeonGreen sequence and the LgBiT (Promega) sequence with a linker sequence (QKAFT-GF-EDFVG). One day before introducing the HiBiT fusion protein, pFT-GeLgBiT was lipofected into 293 cells, and the medium was changed immediately before introducing the fusion protein.

[0059] (3) Genome modification and generation of genome-edited reporter cells 293.RxG (Matsumoto et al., Sci. Rep. 2015, 5, 15325) was used as a reporter cell for genome modification by Cre protein. When Cre protein is introduced into the cell, the sequence between the loxP sequences is removed by Cre, and the downstream GFP gene is expressed.

[0060] Furthermore, we established the F*GxFL reporter cell line, in which the sequence between the loxP sequences is removed by Cre when the Cre protein is introduced into the cells, resulting in expression of the downstream luciferase gene. We modified the vector pLV.SGb.PtR (Kato et al., J. Biochem., 2010, 147(1), 63-71) with a bidirectional promoter to construct the reporter vector pLV.PRLb.F*GxFL, which contains a cassette in which the blasticidin-resistant gene-fused Renilla luciferase is located downstream of the PGK promoter, and a cassette in which loxP, GFP, loxP, and Firefly luciferase are located downstream of the EFS promoter (SEQ ID NO: 13). We introduced this viral vector into HEK293 cells, HeLa cells, HT1080 cells, MCF7 cells, Jurkat cells, and K562 cells, and obtained a cell group that became blasticidin-resistant for each cell line, thereby obtaining a reporter cell line.

[0061] Reporter cells for genome editing by ZFN proteins 293.E2mEme(ZFRR6+2)rald were generated by incorporating a reporter gene (SEQ ID NO: 14) into FlpIn293 cells (Thermo Fisher). This reporter gene has a target site for ZFN proteins incorporated in the middle of the GFP gene downstream of the CMV promoter, and the insertion of the target site causes a frameshift of +2 bases. When the ZFN protein is incorporated into the cell, the target site is cleaved by the ZFN protein, and when a frameshift mutation with a (3n+2) base deletion occurs as a result of repair after this cleavage, the full-length GFP gene is expressed. The target site consists of two ZF recognition sequences (CTTTTGCAGTTT (SEQ ID NO: 15) and AAACTGCAAAAG (SEQ ID NO: 16)) arranged in a palindrome, and is cleaved by a homodimer of one type of ZFN protein.

[0062] The reporter cell 293.E2mEme(F281)rald for genome editing by Cas9 protein was created by incorporating a reporter gene (SEQ ID NO: 17) into FlpIn293 cells (Thermo Fisher). This reporter gene has a target site for the CRISPR-Cas9 system incorporated into the middle of the GFP gene downstream of the CMV promoter, and a +1 base frameshift occurs due to the insertion of the target site. When the CRISPR-Cas9 system is incorporated into the cell, the target site is cut, and when a frameshift mutation with a (3n+1) base deletion occurs as a result of repair after this cut, the full-length GFP gene is expressed.

[0063] (4) Protein introduction The cultured cells were cultured in a CO2 incubator at 37°C in DMEM medium containing 10% serum until just before protein introduction. Just before protein introduction, the DMEM medium containing 10% serum was removed, washed once with a specific medium (described below), and then the protein was added together with the specific medium. A few hours after the addition of the protein, the medium was replaced with DMEM medium containing 10% serum, and cultured for several days. Analysis was performed by flow cytometry (BD Biosciences, Accuri C6) two days after addition in the case of Cre protein, three days after addition in the case of ZFN protein, and three days after addition in the case of CRISPR-Cas9 system introduction.

[0064] The composition of DMEM(+Mg, +Ca) is as follows (Table 1). In addition, DMEM (-Mg, +Ca), DMEM(+Mg, -Ca) and DMEM(-Mg, -Ca)) with the same composition except for excluding calcium and / or magnesium from the composition below were specially ordered and prepared from Nacalai Tesque, Inc.

[0065] [Table 1]

[0066] (5) Luciferase assembly analysis One day before protein transfection, 0.2 μg of pFT-GeLgBiT was lipofected into 293 cells seeded in a 96-well plate using TransIt-293 reagent (Takara Bio), and the medium was replaced just before transfection with HiBiT-fused Cre protein. Regular DMEM or Ca-free DMEM (DMEM (+Mg, -Ca), special order by Nacalai Tesque) was used as the medium. HiBiT-fused Cre protein was added at 1 μM, and luminescence was measured using the Nano-Glo Live Cell Assay System (Promega) and a plate reader (TECAN, Infinite M200 Pro).

[0067] (6) Transcriptional activation analysis One day before protein transfection, 0.1 μg of pG5-Luc (Promega) and 0.01 μg of pRL-CMV (Promega) were lipofected into 293 cells seeded in a 96-well plate, and the medium was replaced just before transfection with Gal4-VP16 protein. The medium used was normal DMEM or Ca-free DMEM (DMEM (+Mg, -Ca), a special order from Nacalai Tesque). Gal4-VP16 protein was added at concentrations of 0.1 μM, 1 μM, and 10 μM, and transcriptional activation was analyzed by measuring luciferase luminescence using the Dual-Glo Luciferase Assay System (Promega) two days later.

[0068] B. Results (1) Effect of calcium ions on Cre protein transfection efficiency In early experiments, the inventors discovered that Cre protein was introduced with high efficiency in HBS buffer (HEPES-buffered saline) rather than in a normal culture medium (Dulbecco's modified Eagle medium: DMEM) (Fig. 1B, C: compare HBS and DMEM-intact). Since DMEM medium contains components such as glucose as a carbon source, amino acids as a nitrogen source, and vitamins in addition to the pH buffer, they hypothesized that the difference in components from HBS was the cause of the difference in activity, and conducted various studies.

[0069] Surprisingly, the addition of EDTA or EGTA, which are divalent metal ion chelators, to the DMEM medium promoted the introduction of Cre protein into the nucleus by more than 10 times (Figure 1C). EDTA and EGTA are not introduced into the cytosol and are known to be reagents with low cytotoxicity.

[0070] Next, we focused on calcium ions and magnesium ions among the divalent metal ions contained in DMEM, and examined whether the introduction of Cre protein would be promoted when either or both of them were removed. In this study, we used a Cre protein (HNCre) in which a His tag and NLS were fused, HCre containing only a His tag and no NLS sequence, and HN[X#A]Cre in which a mutation was introduced into the NLS sequence (PKKKRKV, SEQ ID NO: 18) in HNCre (X indicates the amino acid before modification, # indicates the amino acid position in the NLS, and A indicates a mutation to an alanine residue. For example, N[P1A] means a mutated NLS sequence in which the first P in the NLS sequence is replaced with A).

[0071] As a result, we found that the efficiency of protein introduction was low in normal DMEM medium containing calcium ions and magnesium ions, and that the efficiency of protein introduction improved when calcium ions were removed (Figure 1D). On the other hand, the presence or absence of magnesium ions did not affect the efficiency of protein introduction. Removal of calcium ions improved Cre recombinase activity by approximately 3 to 10 times. Since no such improvement in Cre recombinase activity was observed when plasmid DNA expressing the Cre protein was introduced, it was found that the improvement in Cre recombinase activity by removal of calcium ions was due to the promotion of introduction of the Cre protein into cells.

[0072] It is known that the addition of an NLS sequence improves Cre recombinase activity, but this is not thought to be due to an improvement in nuclear localization activity. This is because the NLS[K3A] mutant has a nuclear localization activity reduced by about 100 times (Hodel et al., J. Biol. Chem., 2001, 276(2):1317-1325), but the Cre recombinase activity in this example is not significantly affected. This fact indicates that the NLS sequence itself functions as a strong cell membrane penetrating peptide (CPP).

[0073] (2) Effect of calcium ions on cell membrane-permeable peptide-dependent protein delivery The above results suggested that the removal of calcium ions in DMEM promoted the function of the nuclear localization signal as a cell membrane-permeable peptide, so other cell membrane-permeable peptides were also examined. In addition to HCre and HNCre, HTCre protein fused with a His tag and Tat peptide, and HTNCre protein fused with a His tag, Tat peptide, and NLS were also examined at various protein concentrations.

[0074] As a result, it was found that the removal of calcium ions improved the efficiency of protein introduction for any protein fused to a cell membrane-penetrating peptide (Figure 2).

[0075] (3) Verification of uptake into the cytosol In the above example, the effect of calcium ions was evaluated by using the introduction of proteins into the nucleus and the recombination of genomic DNA sequences by Cre protein as an index. In this example, a split-luciferase complementation assay was performed to focus on the uptake of proteins into the cytosol. In this evaluation system, a vector expressing LgBiT, a large fragment of split NLuc, is introduced into cells in advance to localize the LgBiT protein in the cytosol. This protein is not released into the medium under normal conditions. When a Cre protein (HNBCre) fused with HiBiT peptide (B), a small fragment of split NLuc, and furimazine, a substrate of NLuc, are added to the medium and introduced into the cells, LgBiT and HNBCre associate with each other in the cells, restoring the function of NLuc and catalyzing the luminescence reaction of furimazine. Luminescence was detected in real time by a microplate reader. In addition, when a nuclear localization signal was fused to LgBiT, the fusion protein was transported into the nucleus, and luminescence was detected in the nucleus. In these evaluation systems, the calcium ion concentration in the medium (DMEM) was changed and its effect was examined (Figure 3A).

[0076] As a result, in cells in which LgBiT was localized in the cytosol in the presence of 2 mM calcium ions contained in normal DMEM, a slight increase in luminescence was observed immediately after HNBCre was introduced, but it soon decreased (Figure 3B). When the calcium ion concentration of the medium was decreased, a large peak in luminescence was observed around 180 minutes when a medium containing 0.2 mM or less calcium ions was used, confirming that the uptake of HNBCre into the cells was promoted (Figure 3B). Similarly, in cells in which LgBiT fused to a nuclear localization signal was introduced, when a medium containing 0.2 mM or less calcium ions was used, luminescence increased after 180 minutes, confirming that the uptake of HNBCre into the cells was promoted (Figure 3C). These facts indicate that HNBCre passes through the cytosol and reaches the nucleus more efficiently when a medium with a low calcium ion concentration is used.

[0077] To verify the correlation between the split luciferase complementation evaluation and the actual Cre recombinase activity, we introduced LgBiT protein and HNBCre protein into reporter cells (293.RxG) for genome modification by Cre protein, and evaluated Cre recombinase activity. As a result, Cre recombinase activity was correlated with split luciferase complementation evaluation (efficiency of Cre protein reaching the nucleus), and Cre recombinase activity was improved when a medium with a low calcium ion concentration was used (Figure 3D, E).

[0078] Next, to examine the effect of the position at which the HiBiT peptide is fused, we similarly examined HBCre, HBNCre, HBTNCre, HNBCre, and HTNBCre, in which the HiBiT peptide was fused to the N-terminus, and HCreB, HNCreB, and HTNCreB, in which the HiBiT peptide was fused to the C-terminus (Figure 3F).

[0079] As a result, in both cases, removal of calcium ions from the medium promoted the import of the protein into the cytosol (Fig. 3G, H, I, J). However, this experiment should be interpreted with caution, since it was observed that Tat's activity as a cell membrane-permeable peptide was lost when the Tat peptide sequence was fused. In fact, when Cre recombinase activity was observed in parallel, it was shown that the CPP activity of Tat was reduced in HiBiT-fused Cre, regardless of the position of HiBiT (Fig. 3K, L).

[0080] Taken together, these results support the idea that the efficiency of protein introduction was improved by lowering the calcium ion concentration in the medium.

[0081] (4) Generality of the introduced protein The introduction of proteins other than Cre into cells was analyzed. First, the introduction of ZFN proteins into cells was examined. Reporter cells were prepared in which the target sequence of the ZFN protein was inserted into the loop portion of GFP. In these reporter cells, GFP was expressed and fluorescence was detected only when the target sequence was cleaved by the ZFN protein and subsequently repaired, resulting in a (3n-2) indel mutation. The results are shown in Figure 4A. As in the results of our previous research, the ZFN proteins were introduced into cells, but in a medium from which calcium ions had been removed (DMEM, -Ca 2+ ) showed improved genome editing activity. On the other hand, the improvement in genome editing activity by adding EDTA to a normal medium (DMEM, +EDTA) was limited, suggesting that EDTA may inhibit the structure formation of ZFN proteins that require zinc ions.

[0082] Similarly, indel reporter cells were prepared for Cas9 to evaluate the effect of calcium ions on protein introduction. The Cas9 protein was tagged with Tat peptide, 3xNLS, and His tag. In vitro, a complex was formed between the Cas9 protein and guide RNA, and this complex was added directly to the medium. In this case, it was found that the introduction efficiency improved when the Cas9 protein was in the range of 0.5 μM to 10 μM when calcium ions were removed from the medium (Figure 4B).

[0083] The above examples were mainly verifications in an experimental system in which mutations caused by transient genome modification are inherited by dividing daughter cells. Introduction of proteins into cells was also verified in an experimental system using transcription activation as an indicator. The DNA binding domain Gal4 specifically binds to the UAS sequence, and can activate transcription of the adjacent minimal promoter by fusing with the VP16 transcription activation domain. The present inventors prepared cells into which pG5-Luc carrying the Firefly luciferase gene was introduced downstream of the UAS, and added purified His tag, nuclear localization signal, and Gal4-VP16 fusion protein to these cells. As a result, improved transcription activation was observed when calcium ions were not added (Figure 4C).

[0084] (5) Generality of protein-transfected cells Using the F*GxFL reporter cell line described in A(3) above, the efficiency of protein introduction under low calcium ion concentration conditions was compared. In these cell lines, Renilla luciferase is expressed under the control of the PGK promoter, and GFP is expressed under the control of the EFS promoter. When the Cre protein is introduced into the cells and recombination occurs at the loxP site, Firefly luciferase is expressed instead of GFP. The efficiency of Cre protein introduction was evaluated as a relative value obtained by dividing the measured value of Firefly luciferase by the measured value of Renilla luciferase.

[0085] The results are shown in Figures 5A to 5F. In all cell lines, the efficiency of protein introduction was improved under low calcium ion concentration conditions. An improvement in protein introduction efficiency was observed under calcium ion concentration conditions of 0.5 mM or less, and a particularly significant improvement in protein introduction efficiency was observed under calcium ion concentration conditions of 0.2 mM or less. These results demonstrated that proteins can be introduced with high efficiency under low calcium ion concentration conditions, regardless of the tissue from which the cells originate and whether they are adherent cells or suspension cells.

[0086] (6) Cytotoxicity evaluation of calcium ion-depleted medium Generally, the efficiency of substance introduction into cells is in a trade-off relationship with cytotoxicity, and cytotoxicity tends to increase under conditions where more protein can be introduced. Buffer solutions such as HBS used in conventional protein introduction methods consist only of pH buffers and salts, and do not contain nutrients necessary for cell growth, which may hinder cell growth. Therefore, HEK293_F*GxFL cell lines were cultured for 1 hour in buffer solutions or media containing or not containing FBS, and then Renilla luciferase luminescence was measured using the Dual-Glo Luciferase Assay System. If cell activity is reduced and / or cell growth is inhibited, the Renilla luciferase luminescence value will decrease.

[0087] The results are shown in Figures 6A and 6B. The vertical axis of the graph indicates the luminescence value of Renilla luciferase. The activity of cells cultured in HBS buffer and PBS buffer decreased, whereas the activity of cells cultured in DMEM medium with low calcium ion concentration (DMEMΔCa, DMEM+EDTA, DMEM+EGTA) was maintained. Furthermore, no difference was observed depending on the presence or absence of FBS. This result suggests that the protein introduction method using a medium with only a reduced calcium ion concentration has less negative effects on cells.

[0088] (7) Whether or not the His tag affects protein delivery Since all of the proteins used above were His-tagged, to exclude the possibility that the His-tag might affect the protein introduction efficiency, we performed the same test as above using Cre(ΔHis) and NCre(ΔHis), which do not contain a His-tag.

[0089] The results are shown in Figure 7. The vertical axis of the graph indicates the ratio of GFP-positive cells (100% of cells are GFP-positive is set as 1). Regardless of the presence or absence of the His tag, the efficiency of protein introduction was improved under low calcium ion concentration conditions.

[0090] The present inventors found that the efficiency of protein introduction into animal cells was dramatically improved by removing calcium ions, which are usually contained in the culture medium and extracellular fluid of cultured animal cells. This efficient protein introduction method using this phenomenon was a versatile method that could be applied to genome modification using Cre protein, genome editing using ZFN and CRISPR-Cas9, transcriptional control by Gal4-VP16, and complementation evaluation using split luciferase. [Industrial Applicability]

[0091] As described above, according to the present invention, it is possible to dramatically improve the efficiency of introducing proteins into animal cells. The method of the present invention is simple and does not require special reagents or devices to introduce proteins into cells, and has little negative effect on the activity and characteristics of the cells obtained. In addition, it is versatile because it does not matter what type of protein is introduced. Therefore, in addition to being used in basic research, it can be used in various industrial fields including the medical field depending on the type of protein and animal cell to be introduced.

Claims

1. A method for introducing a protein that does not contain a protein transduction domain into an animal cell by protein transduction, the method comprising the step of contacting the protein that does not contain a protein transduction domain with an animal cell in a medium having a calcium ion concentration of 0 to 0.3 mM.

2. 2. The method of claim 1, wherein the calcium ion concentration is 0.1 to 0.2 mM.

3. The method according to claim 1 or 2, wherein the magnesium ion concentration in the medium is 0 to 2 mM.

4. The method according to any one of claims 1 to 3, wherein the medium contains a calcium chelating agent.

5. 5. The method of claim 4, wherein the calcium chelating agent is selected from the group consisting of EDTA, EGTA and BAPTA.

6. A medium for introducing a protein not containing a protein transduction domain into an animal cell by protein transduction, the medium having a calcium ion concentration of 0 to 0.3 mM and a magnesium ion concentration of 0 to 2 mM.

7. The medium according to claim 6, wherein the calcium ion concentration is 0.1 to 0.2 mM.

8. The medium according to claim 6 or 7, further comprising a calcium chelating agent.

9. 9. The medium of claim 8, wherein the calcium chelating agent is selected from the group consisting of EDTA, EGTA and BAPTA.

10. A composition for promoting the introduction of a protein that does not contain a protein transduction domain into an animal cell by protein transduction, comprising a calcium chelator.

11. 11. The composition of claim 10, wherein the calcium chelating agent is selected from the group consisting of EDTA, EGTA and BAPTA.

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