JP5097412B2 - Protein intracellular introduction agent - Google Patents

Description

  The present invention relates to a novel conjugate of polyethyleneimine or a chemically modified derivative thereof and glutathione, an introduction agent for introducing a protein into a cell using the conjugate, and a method thereof.

  In order to biologically function an arbitrary protein in a cell, it is preferable to introduce the protein itself into the cell. However, the cell membrane is composed of a lipid bilayer, and a large foreign molecule such as a protein is excluded. It is not easy to penetrate into cells.

  The present inventors have previously developed a method for introducing a protein into a living cell through electrostatic adsorption to the surface of a living cell having a negative charge by cationizing the protein. . The method uses a specific bond such as antibody-Protein A / G or biotin-avidin (Patent Document 1), and a method for intracellularly introducing a protein or peptide using polyethyleneimine (PEI) (Patent Document 2). By these methods, various proteins prepared in advance outside the cell can be easily introduced into living cells.

  Specifically, the method for introducing a protein or peptide into a cell using polyethyleneimine specifically includes, for example, converting EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) into a protein or peptide and polyethyleneimine. , DCC (N, N-dicyclohexylcarbodiimide), SPDP (N-succinimidyl-3- (2-pyridyldithio) propionate), 2-iminothiolane, GMBS (N- (4-maleimidobutyryloxy) succinimide), etc. This is a method in which a complex is produced by covalent bonding, and when the complex is added and cultured in a medium containing cells into which the protein is to be introduced, the complex is taken into the cells (Patent Document 2). ).

  However, in this method for introducing a protein or peptide into cells using polyethyleneimine, the target protein or peptide and polyethyleneimine must be covalently bound using a reagent such as EDC.

In addition, a conventional method of introducing a protein into a cell, a method of fusing a basic peptide (Non-patent Document 1), and a method of introducing a complex with the cationized avidin (Patent Document) Although there is 1), these methods are inferior in convenience. Various protein introduction reagents (Chariot ^™ , Bioporter ^™, etc.) based on cationic lipids are also commercially available, but the introduction efficiency is not always good.

  On the other hand, glutathione S transferase (GST) fusion proteins capable of soluble expression in Escherichia coli and simple affinity purification as recombinant proteins have been widely used all over the world since the 1990s, and are widely used in basic research fields. Recently, it has been reported that the GST fusion protein itself is well taken up into cells (Non-patent Document 2). However, according to the results of the inventors' additional tests, the introduction efficiency was very low.

Japanese Patent Application No. 2002-342486 JP 2004-49214 A Schwarze SR, Ho A, Vocero-Akbani A and Dowdy SF. , In vivo protein transduction: delivery of a biologically active protein into the mouse. , Science, 285 (5433), 1569-1572, 1999. Namiki S, Tomida T, Tanabe M, Iino M and Hirose K, Intracellular delivery of glutathione S-transferred into mammalian cells. Biochem. Biophys. Res. Commun. 305 (3), 592-597, 2003.

  An object of the present invention is to provide a reagent and a method for introducing a glutathione S transferase fusion protein into cells with high efficiency by a simple operation.

  The present inventors used polyethyleneimine or a chemically modified derivative thereof so that a fusion protein of glutathione S transferase and a target protein (hereinafter simply referred to as “glutathione S transferase fusion protein”) can be efficiently introduced into cells. The cationization method was applied to glutathione S transferase fusion protein. The inventors have succeeded in synthesizing a novel conjugate of polyethyleneimine or a chemically modified derivative thereof and glutathione for use in this application. By using this conjugate, it was found that proteins can be efficiently introduced into cells by a simple operation, and the present invention was completed.

The features of the present invention are summarized as follows.
(1) A conjugate of polyethyleneimine or a chemically modified derivative thereof and glutathione. Preferably, polyethyleneimine and glutathione are in a molar ratio of 1: 1 to 1:20, more preferably 1: 1 to 1:10, still more preferably 1: 1 to 1: 5, and particularly preferably 1: 1 to 1. : Conjugate bound at 3.
(2) The conjugate according to (1), wherein the polyethyleneimine has a number average molecular weight in the range of 100 to 100,000.
(3) The conjugate according to (1) or (2), wherein the polyethyleneimine has a branched structure.
(4) The conjugate according to any one of (1) to (3), wherein the chemically modified derivative is an N-alkyl derivative, N-acyl derivative, N-sulfonyl derivative or Schiff base derivative of polyethyleneimine.
(5) The conjugate according to any one of (1) to (4), wherein the glutathione is a reduced glutathione.

(6) The conjugate according to any one of (1) to (5), wherein polyethyleneimine and glutathione are bonded via a disulfide bond.
(7) A complex of the conjugate according to any one of (1) to (6) and a glutathione S transferase fusion protein.
(8) The complex according to (7), wherein the conjugate and the glutathione S-transferase fusion protein are bound at a molar ratio of 1:10 to 100: 1.
(9) Polyethyleneimine or a chemically modified derivative thereof and 2,2-dipyridyl disulfide are reacted in the presence of a reagent that reacts with the amino group of polyethyleneimine or a chemically modified derivative thereof to expose a thiol group. A method for producing a conjugate according to any one of (1) to (6), comprising a step of bringing reduced glutathione into contact with a product.
(10) The method according to (9), wherein the reagent is γ-thiobutyrolactone, methyl 4-mercaptoalkaneimidate, 2-iminothiolane or N-acetylhomocysteine thiolactone.

(11) The method according to (9) or (10), wherein the chemically modified derivative is an N-alkyl derivative, N-acyl derivative, N-sulfonyl derivative or Schiff base derivative of polyethyleneimine.
(12) The method according to (7) or (8), comprising mixing the conjugate according to any one of (1) to (6) and a glutathione S-transferase fusion protein to form a complex thereof. A method for producing a composite.
(13) The method according to (12), comprising mixing the conjugate and glutathione S-transferase fusion protein in a molar ratio of 1: 1 to 100: 1.
(14) The method further comprises the step of preparing a fusion gene by fusing a gene encoding glutathione S transferase and a gene encoding the target protein, and expressing the fusion gene in E. coli to obtain a glutathione S transferase fusion protein. ) Or the method according to (13).
(15) A method for introducing a protein into a cell, comprising transporting the target protein into the cell using the conjugate according to any one of (1) to (6).

(16) The method according to (15), comprising mixing the conjugate and glutathione S-transferase fusion protein to form a complex thereof.
(17) The method according to (16), wherein the mixture ratio of the conjugate and glutathione S-transferase fusion protein is 1: 1 to 100: 1 in terms of molar ratio.
(18) A method for introducing a protein of interest into a cell, comprising transporting the protein into a cell using the complex according to (7) or (8).
(19) A protein intracellular introduction agent comprising the conjugate according to any one of (1) to (6).
(20) A protein intracellular introduction agent comprising the complex according to (7) or (8).
(21) A protein intracellular introduction kit comprising the protein intracellular introduction agent according to (19).
(22) A protein intracellular introduction kit comprising the protein intracellular introduction agent according to (20).

  Hereinafter, in this specification, polyethyleneimine may be expressed as PEI, glutathione as GSH, and glutathione S transferase as GST.

  Moreover, the description of a conjugate | bonded_body and a composite_body | complex connects the component of a conjugate | bonded_body or a composite_body | complex with "-", and shows that the components are couple | bonded in the connected order. For example, the “PEI-GSH-GST fusion protein” complex is composed of polyethyleneimine, glutathione, glutathione S transferase fusion protein, and indicates a complex bound in this order.

  According to the present invention, a glutathione S-transferase fusion protein can be easily and efficiently introduced into cells by using a conjugate of polyethyleneimine or a chemically modified derivative thereof and glutathione.

  The conjugate of the present invention is a coupling product of polyethyleneimine or a chemically modified derivative thereof and glutathione.

  Polyethyleneimine (PEI) may have a linear or branched structure, and the branched structure includes a bridged structure. The branched chain PEI is represented by the following formula, for example.

（式中、ｘ及びｙはそれぞれ１以上の整数である。）

(In the formula, x and y are each an integer of 1 or more.)

  In the above formula, the amino moiety at the end of the branched chain may be a free amino group or represents a further polymer chain of ethyleneimine.

  PEI is a water-soluble polymer with a large positive charge density. PEI is also used as a food additive such as kamaboko precipitant, and its safety to the living body has been confirmed.

  PEI preferably has a branched chain structure, for example, the following formula

PEI having a branched structure as exemplified in the above is preferable because of higher positive charge density. Further, the molecular weight is preferably PEI having a molecular weight in the range of 100 to 100,000, more preferably 100 to 10,000, and even more preferably 200 to 3,000 in consideration of cell introduction efficiency, handleability, and the like. PEI having a low molecular weight of 500 to 1,800 is particularly preferable.

  PEI is commercially available. For example, Epomin (trademark) manufactured by Nippon Shokubai Co., Ltd. is preferably used.

  In addition, a chemically modified derivative of PEI can be formed from an amino group (preferably a primary amino group) of PEI and all chemical modifiers that can react with the amino group. Examples of chemical modifiers include substituted or unsubstituted, saturated or unsaturated, aliphatic, aromatic, alicyclic and heterocyclic alkylating agents, acylating agents, sulfonylating agents, etc. .

  As used herein, “aliphatic” refers to an alkyl group, an alkenyl group, or an alkynyl group, preferably having 1 to 6 carbon atoms (C1 to C6), more preferably 1 to 4 carbon atoms (C1 to C4). ) Hydrocarbon groups such as methyl, ethyl, propyl, butyl and the like.

  As used herein, “aromatic” refers to a hydrocarbon group containing a benzene nucleus, and examples thereof include a phenyl group and a phenylene group.

  As used herein, “alicyclic” refers to a saturated monocyclic hydrogen group or an unsaturated monocyclic hydrocarbon group containing one or two double bonds, such as a cyclopentyl group, a cyclohexyl group, A cyclohexenyl group etc. are mentioned.

  As used herein, “heterocyclic” refers to a monocyclic or fused cyclic hydrocarbon group containing one or more heteroatoms (eg, N, O, S, etc.) in a saturated or unsaturated ring. Pyrrolidyl, pyridyl, piperidinyl, piperazinyl, morpholino, indolyl, imidazolyl, thiophenyl and the like.

  Examples of the substituent include a halogen atom (F, Cl, Br or I), phenyl, hydroxy, amino, cyano, alkoxy, alkylamino, dialkylamino, carbonyl, alkoxycarbonyl and the like.

Examples of alkylating agents are alkyl halides, epoxides and the like.
Examples of acylating agents are esters, acid halides, acid anhydrides and the like.
Examples of sulfonylating agents are alkylsulfonyl halides and the like.

  Another example is an aldehyde, which forms a Schiff base by reaction with an amino group.

  Examples of specific alkylating agents include styrene oxide, epoxybutene, halogenated C1-C6 alkyl, preferably C1-C4 alkyl, C1-C4 alkenyl such as methyl, ethyl, propyl, butyl, propenyl, butenyl and the like. It is done.

  Specific examples of acylating agents include C1-C6 alkanoyl halides, preferably C1-C4 alkanoyl halides such as acetyl chloride, propionyl chloride, butyryl chloride, and the like.

  Specific examples of the sulfonylating agent include C1-C6 alkanesulfonyl halides, preferably C1-C4 alkanesulfonyl halides such as methanesulfonyl chloride and ethanesulfonyl chloride.

Accordingly, in the present invention, chemically modified derivatives of PEI include, for example, N-alkyl derivatives, N-acyl derivatives, N-sulfonyl derivatives, Schiff base derivatives, and the like.
Glutathione (GSH) is represented by the following formula.

  Glutathione is a tripeptide in which glutamic acid, cysteine, and glycine are peptide-bonded in this order. However, since the bond between glutamic acid and cysteine is a γ-glutamyl bond consisting of the γ-carboxy group of the glutamic acid side chain and the α-amino acid of the cysteine main chain, it is resistant to most proteases and is not degraded, and α-glutamyltrans Only a limited number of enzymes such as peptidase can be degraded.

  Glutathione is present at a concentration of 0.5 to 10 mM in cells of various biological species, regulates cell functions by scavenging radicals and redox, and is also an SH donor for various enzymes. Also known as an antioxidant component, supplements containing glutathione are commercially available.

  Glutathione has a reduced form (GSH) and an oxidized form (GSSG), and the oxidized form is a molecule in which two molecules of reduced glutathione are bonded by a disulfide bond. Most intracellular glutathione is reduced. In the present invention, it is preferable to use reduced glutathione. In the present specification, unless otherwise specified, glutathione indicates reduced glutathione (GSH).

  Glutathione is commercially available, and for example, GSH from Nacalai Tesque, Inc., Glutathione reduced (GSH) from Roche Applied Science, etc. are preferably used.

  In the conjugate of the present invention, PEI and GSH are bonded via a disulfide bond, and the molar ratio is preferably 1: 1 to 1:20, more preferably 1: 1 to 1:10, and still more preferably 1: The bonds are in the range of 1 to 1: 5, particularly preferably 1: 1 to 1: 3.

  Furthermore, a glutathione S transferase fusion protein (GST fusion protein) can be further bound to the PEI-GSH conjugate of the present invention.

  The GST fusion protein is a fusion of the GST and a target protein to be introduced into a cell. GST is an enzyme that is widely present in various biological species and transfers glutathione to various compounds such as drugs to produce glutathione conjugates. The target protein is not particularly limited, and includes any protein such as a peptide, an enzyme, and other proteins having functionality (physiological activity or biological activity). It also includes proteins to which chemical modification groups such as sugar chains, lipids, and phosphate groups are bound. Furthermore, the protein may be in its native state or in a denatured state. Examples of the target protein include p53, Caspase, PPARγ, and artificially modified protein.

  The conjugate of the present invention and the GST fusion protein bind preferably in a molar ratio of about 1:10 to 100: 1, more preferably 1: 5 to 10: 1, and even more preferably 1: 2 to 2: 1. ing. For the binding mode of GST and glutathione, see Nishida M, Harada S, Noguchi S, Satow Y, Inoue H and Takahashi K. et al. , Three-dimensional structure of Escherichia coli glutathione S-transfer complex with glutensulfate of Cys. , J .; Mol. Biol. , 281 (1), 135-147, 1998, as is clear from the results of X-ray crystal structure analysis of the complex, one molecule of glutathione molecule binds to one molecule of GST protein.

Hereinafter, the manufacturing method of the conjugate | bonded_body of this invention is demonstrated.
The method for producing a conjugate of the present invention comprises reacting PEI or a chemically modified derivative thereof with 2,2-dipyridyl disulfide in the presence of a reagent that reacts with the amino group of PEI or a chemically modified derivative thereof to expose a thiol group. And a step of bringing reduced glutathione into contact with the obtained product.

For example, polyethyleneimine having a number average molecular weight of 100 to 100,000 can be suitably used. For example, Epomin (number average molecular weight 300, 600, 1,200, 1,800, 10, 000, 70,000) can be used.
2,2-dipyridyl disulfide is represented by the following formula.

  As 2,2-dipyridyl disulfide, for example, Aldrithiol (trademark) manufactured by Sigma or 2-PDS manufactured by DOJINDO can be preferably used.

  Examples of the reagent that exposes the thiol group by reacting with the amino group of polyethyleneimine or a chemically modified derivative thereof include γ-thiobutyrolactone, methyl 4-mercaptoalkaneimidate, 2-iminothiolane, or N-acetylhomocysteine thiolactone. . In particular, γ-thiobutyrolactone is suitable, and γ-thiobutyrolactone is represented by the following formula.

  As γ-thiobutyrolactone, for example, γ-thiobutyrolactone manufactured by SIGMA-ALDLICH can be suitably used.

  The PEI-GSH conjugate of the present invention can be synthesized, for example, according to the scheme of FIG. Specifically, for example, it can be synthesized by the following procedure.

  First, PEI is dissolved in a solvent such as tetrahydrofuran (THF), and aldolthiol is dissolved therein. Γ-thiobutyrolactone is added thereto with stirring, and the mixture is stirred for several hours at about 50 ° C., and then the supernatant separated into two layers is concentrated by a rotary evaporator. This is redissolved with ultrapure water and washed with hexane, and then about 0.9 times the molar equivalent of reduced glutathione to the aldolthiol used in the reaction is added to obtain a PEI-GSH conjugate. .

  Alternatively, PEI can be chemically modified as described above to synthesize a modified PEI-GSH conjugate. For example, PEI is dissolved in a suitable solvent, a chemical modifier that reacts with the amino group of PEI is added, and the reaction is carried out for about several hours at about 0 to several tens of degrees Celsius in the presence of a base such as an amine as necessary. , Make the modification. Using the obtained modified PEI and GSH, a modified PEI-GSH conjugate is synthesized in the same manner as the above synthesis procedure.

  The method may further comprise contacting the GST fusion protein with the PEI-GSH conjugate or modified PEI-GSH conjugate synthesized as described above. By this step, GSH and GST are specifically bound to form a PEI-GSH-GST fusion protein or a modified PEI-GSH-GST fusion protein complex. The molar ratio of PEI-GSH conjugate or modified PEI-GSH conjugate to GST fusion protein upon contact is preferably 1: 1 to 100: 1, more preferably 1: 1 to 20: 1, most preferably 1. : 1 to 10: 1.

  The GST fusion protein is prepared, for example, by fusing a gene encoding GST and a gene encoding a target protein, and integrating the gene into an appropriate expression vector. It can be obtained by expressing in a host cell such as an animal cell. Preferably, GST gene fusion system, which is widely used as one of effective expression methods for recombinant proteins, is used. The advantages of this system are that it is easy to express proteins as soluble, and that it can be easily purified using affinity chromatography techniques using the GST moiety, and is widely used all over the world. It is a technique. The GST fusion protein obtained here is mainly used for various biochemical experiments in vitro (external to living cells).

  Expression vectors used in the GST gene fusion system are commercially available as kits, such as the pGEX series (GE Healthcare Biosciences, Amersham Pharmacia) and the pET series (Novagen). Hereinafter, as an example, a method for producing a GST fusion protein using a pGEX vector will be described.

The pGEX vector is a vector in which a protease recognition site and a multiple cloning site are arranged downstream of the GST gene sequence. The target protein gene can be incorporated in a frame downstream of the GST gene (eg, from Schistosoma japonicum) on the vector, and expressed in a large amount as a fusion protein in E. coli cells. The pGEX vector has a lac I ^q gene, and can induce protein expression by adding IPTG (isopropyl-1-thio-β-galactoside), and can use various E. coli as hosts. it can. For example, E. coli such as BL21, DH5α, JM109, TG1, and XL1 Blue can be suitably used. A PCR amplified fragment to which a recognition sequence of a restriction enzyme (eg, Eco RI, Xhol, SacI, SmaI, BamHI, AccI, PstI, HindIII, etc.) is added using the target protein cDNA as a template is ligated to pGEX, Is made. This is transformed into the above-mentioned Escherichia coli and cultured at 37 ° C. in a medium containing ampicillin (LB (Amp ⁺ ) or the like), for example. O. D. When 600 = 0.6 to 0.8, IPTG is added to a final concentration of, for example, 0.1 to 1 mM, and cultured at 30 ° C. for 2 to 5 hours, for example. It should be noted that the optimum conditions for the IPTG concentration, the culture temperature, and the culture time differ depending on the target protein, so it is preferable to examine them in advance. Bacteria are collected by centrifugation, suspended in a buffer (PBS, GST buffer, etc.), and the cells are destroyed using, for example, a sonicator. Centrifugation is performed, and the supernatant is collected as bacterial protein. For example, a small amount of equilibrated glutathione-Sepharose 4B is added, and gently shaken at 4 ° C. for 30 minutes to 2 hours. Centrifuge at 4 ° C., discard the supernatant, and collect glutathione-Sepharose 4B. Glutathione-Sepharose 4B is washed repeatedly with buffer. Here, the purified sample, the soluble fraction after sonication, the insoluble fraction, and the glutathione-sepharose supernatant after adsorption are electrophoresed by SDS-PAGE to confirm purification.

  After purification, glutathione-sepharose 4B can be eluted using affinity beads. Glutathione-Sepharose 4B is centrifuged to remove the supernatant, and the same amount of elution buffer as the resin is added. For example, the mixture is gently stirred at 4 ° C. for 5 to 10 minutes and centrifuged to collect the supernatant several times. Collect the GST fusion protein.

Hereinafter, the protein intracellular introduction method of the present invention will be described.
The protein intracellular introduction method of the present invention uses a PEI-GSH conjugate or a modified PEI-GSH conjugate. When the PEI-GSH conjugate or the modified PEI-GSH conjugate obtained by the above production method and the GST fusion protein are mixed in advance, for example, in a test tube, GSH and GST are specifically bound, and the PEI-GSH-GST fusion is performed. A protein complex or a modified PEI-GSH-GST fusion protein complex is formed. The protein can be introduced into the cell simply by adding it to the cell culture supernatant, which is very convenient. The complex PEI-GSH conjugate (modified PEI-GSH conjugate): GST fusion protein molar ratio is preferably 1: 1 to 100: 1, more preferably 1: 1 to 20: 1, and even more preferably 1. : 1 to 10: 1, most preferably 10: 1. FIG. 2 schematically shows how the PEI-GSH-GST fusion protein is introduced into living cells.

  Since the present invention uses specific binding between GST and GSH, and only GST fusion proteins present in various mixed solutions can be introduced into cells, the following handling is also possible. That is, it is known that intracellular proteins regulate physiological activities through post-translational modifications such as phosphorylation. For the purpose of verifying these, GST fusion proteins that have been subjected to post-translational modification-like chemical modification by treating the in vitro GST fusion protein with certain enzymes, tissue / cell extracts, compounds, and chemical reaction inducers And adding a PEI-GSH conjugate or a modified PEI-GSH conjugate to this mixed solution to form a complex of the target GST fusion protein and the PEI-GSH conjugate or the modified PEI-GSH conjugate. As a result, only the GST fusion protein present in the mixture can be introduced into the cell. Therefore, it is possible in principle to perform such an experiment without going through a special purification operation.

  As described above, the PEI-GSH conjugate (modified PEI-GSH conjugate) or PEI-GSH-GST fusion protein complex (modified PEI-GSH-GST fusion protein conjugate) of the present invention is a protein intracellular introduction agent. Can be used as In addition to being used as a research reagent, it can also be used as pharmaceuticals, diagnostics, etc. Specifically, cell function analysis by introducing proteins that affect signal transduction and proteins involved in gene expression, and regenerative medicine such as skin and liver by introducing proteins that promote cell growth, It can be used for cancer treatment by introducing a protein that suppresses cell proliferation or induces apoptosis.

  In addition, a protein comprising a PEI-GSH conjugate (modified PEI-GSH conjugate) or a PEI-GSH-GST fusion protein complex (modified PEI-GSH-GST fusion protein complex) as an intracellular introduction agent of the protein. It is also possible to prepare an intracellular introduction kit. Specifically, for example, a kit for cell function analysis by introducing a protein that affects signal transduction or a protein involved in gene expression, or regeneration of skin or liver by introducing a protein that promotes cell proliferation Examples include kits for cancer treatment by introducing a protein that inhibits medical treatment or cell proliferation or induces apoptosis.

  Although there are various techniques for introducing proteins into cells, the present invention is the first example specialized for glutathione S-transferase fusion proteins.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but these examples do not limit the scope of the present invention.

Purification of GST and GST fusion protein GST derived from Schistosoma japonicum and its GST fusion protein contain 100 μg / mL ampililin using E. coli BL21 (DE3) as a host using pGEX vector (GE Healthcare Biosciences) It was expressed in LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl). GST and its GST fusion protein expressed in the microbial cells were applied to glutathione sepharose gel (GE Healthcare Biosciences) after disrupting the microbial cells using an ultrasonic crusher (BRANSON, SONIFIER 250), and PBST ( After washing with PBS containing 10% Tween), elution was performed with 50 mM Tris (pH 8.0) containing 10 mM GSH.

Synthesis of PEI-GSH conjugate The PEI-GSH conjugate was synthesized according to the scheme of FIG. First, 5.0 g of polyethyleneimine having an average molecular weight of 600 (Nippon Shokubai Co., Ltd., Epomin (trademark) SP-006) was dissolved in 20 ml of tetrahydrofuran, and 5.49 g of aldrithiol (Sigma, chemical name: 2,2-dipyridyl disulfide) was dissolved. To this, 2.54 g of γ-thiobutyrolactone (Sigma) was added with stirring, and the mixture was stirred at 50 ° C. for 2 hours, and then the supernatant separated into two layers was concentrated by a rotary evaporator. This was redissolved with ultrapure water and washed with hexane, and then 0.9 times the molar equivalent (6.89 g) of reduced glutathione to the aldolthiol used in the reaction was added to bind PEI-GSH. Acquired the body.

Intracellular introduction of GST using PEI-GSH conjugate Using the PEI600-GSH conjugate prepared in Example 2, intracellular introduction of GST was quantitatively confirmed. Specifically, a Chinese hamster ovary-derived cell (CHO cell) is used as a protein-introducing cell, and examination of a mixing ratio suitable for intracellular introduction of GST and PEI600-GSH conjugate, and PEI600-GSH-GST complex The time-dependent and concentration-dependent uptake of was quantified and verified by Western blotting using an anti-GST antibody (FIG. 3). Specifically, after the cells were seeded and cultured 12-15 hr, medium was replaced with _O PTI -MEM (INVITROGEN Corp.) in a state became 70-80% confluent were incubated for 30 minutes, and PEI600-GSH A mixed solution of GST was added and further cultured for 1 hour. Thereafter, the cells were collected, and the obtained cell lysate was electrophoresed on a 5 to 20% gradient agarose gel, and transferred to a nitrocellulose membrane after electrophoresis. After the transfer, blocking was performed, and reaction with a goat anti-GST antibody (GE Healthcare Bioscience) was performed. Further, it was reacted with an alkaline phosphatase-fused anti-goat IgG antibody (EY Laboratories), light-emitted using CDP-Star (NEW ENGLAND BioLabs.), And exposed to a film for detection.

  As a result, the single GST was hardly taken up into the cell, but when the mixing ratio of GST: PEI600-GSH conjugate was 1:10, it could be taken up into the cell most efficiently. As shown in FIG. 3A (100 nM GST was mixed with 0 μM, 0.1 μM, 1 μM, 10 μM of each PEI600-GSH conjugate, added to the culture supernatant of CHO cells, and the cells were collected after 1 hour. Quantified by Western blotting with the above anti-GST antibody.)).

  In addition, the complex of GST and PEI600-GSH conjugate (mixing ratio 1:10) was rapidly taken up by the cells and observed to be almost saturated 30 minutes after the addition. This suggests that the amount of binding to the cell surface is completed within 30 minutes (FIG. 3B (100 nM GST mixed with 1 μM PEI600-GSH conjugate (1:10 molar ratio)). It was added to the culture supernatant of the cells, and the cells were collected after each time of 0 minutes, 10 minutes, 30 minutes, and 60 minutes, and quantified by Western blotting using the anti-GST antibody)).

  Furthermore, it was confirmed that the PEI-GSH-GST complex (mixing ratio 1:10) increased depending on the concentration added to the culture supernatant (C in FIG. 3 (mixing of GST and PEI600-GSH conjugate). The ratio was fixed at 1:10 (molar ratio), and complexes of 0 μM, 0.01 μM, 0.1 μM, 1 μM, and 10 μM were added to the culture supernatant of CHO cells, and the cells were collected after 1 hour. And quantified by Western blotting with the anti-GST antibody)).

  In this quantitative system using the Western blotting method, GST that remains bound to the cell surface is not distinguished from GST that is taken into the cell. However, since it has been confirmed from other experiments that the efficiency of introducing a protein cationized with PEI into cells is proportional to the amount bound to the cell surface (Murata H, Sakaguchi M, Futami J, Kitazoe M). , Maeda T, Doura H, Kosaka M, Tada H, Seno M, Huh NH and Yamada H., Denatured and reversibly cationized p53 readily enters cells and simultaneously folds to the functional protein in the cells., Biochemistry, 45 (19), 6124-6132, 2006.), this method conforms to this The assumption that, were quantified.

Intracellular introduction of GST-eGFP fusion protein using PEI-GSH conjugate To visualize the incorporation of GST fusion protein into cells using PEI600-GSH conjugate prepared in Example 2, GST was fused. The green fluorescent protein (eGFP) thus prepared was prepared as a model protein in the same manner as in Example 1, and uptake into various cells was performed using a confocal laser microscope (Carl Zeiss / LSM 510 ver. 3.0). Observed. Specifically, cells were seeded on a 12-well plate loaded with a sterilized cover glass and cultured for 12 to 14 hours. The medium in the state of confluence of about 70-80% is replaced with _O PTI -MEM (INVITROGEN Corp.), it was incubated for 30 minutes. Thereafter, a mixed solution of 500 nM PEI600-GSH conjugate and 500 nM GST fusion protein was added, and further cultured for 2 hours. Thereafter, the cell surface was washed with 20 mM DTT to remove GST-eGFP only bound to the cell surface. After washing, the cover glass was taken out and the fluorescence was observed using a confocal laser microscope.

  As a result, two hours after the addition of the conjugate, uniform and efficient GST-eGFP protein was obtained in all four types of cells (CHO cells, Balb / c 3T3 cells, HeLa S3 cells, Cos7 cells) used for evaluation. Uptake was observed (FIGS. 4A-F).

Confirmation of functional expression by introduction of GST-NLS-Cre recombinase into cells After introduction of GST fusion protein into cells using PEI600-GSH conjugate, GST fusion that has reached the cytoplasm mainly through the pathway shown in FIG. A model experiment was conducted to confirm that the protein expressed its physiological function. In this evaluation system, Cre recombinase was used as a protein to be introduced into the cells, and evaluation system cells in which a reporter gene using the Cre / LoxP system shown in FIG. 5A was stably incorporated into CHO cells were used. In addition, since this reporter gene exists in the nucleus of the cell and it is necessary to transfer the protein taken into the cytoplasm further into the nucleus, a GST fusion protein in which a nuclear translocation signal (NLS) and Cre recombinase are fused (GST-NLS-Cre recombinase) was introduced into the cells. When 100 nM GST-NLS-Cre recombinase was added to the cells for evaluation and observed 48 hours later, no change was confirmed (B in FIG. 5). On the other hand, when a complex formed by mixing 1 μM PEI600-GSH conjugate and 100 nM GST-NLS-Crerecombinase was added, cells with Cre recombinase activity expressed intracellularly (intranuclear) 48 hours later (Red fluorescence) was confirmed (C in FIG. 5).

  From the above results, it was confirmed that the GST fusion protein taken into the cytoplasm via the PEI600-GSH conjugate expressed its function in the cell.

Examination of the mixing ratio (binding ratio) of the PEI-GSH conjugate The PEI-GSH conjugate was prepared in the same manner as in Example 2 except that PEI and GSH were mixed at a mixing ratio of 1: 1, 1: 2, and 1: 3. Produced.

  Next, in the same manner as in Example 4, GST-eGFP fusion protein was introduced into cells using the prepared conjugate. As a result, it was found that the PEI-GSH conjugate having a mixing ratio (binding ratio) of 1: 3 had the highest introduction efficiency (FIG. 6, in the case of GST-eGFP alone, the 1: 1 PEI-GSH conjugate was When GST-eGFP is introduced using GST-eGFP, a 1: 3 PEI-GSH conjugate is used to introduce GST-eGFP).

  Further, a PEI-GSH conjugate was obtained in the same manner as in Example 2 except that PEI having an average molecular weight of 1800 was used and the mixing ratio of PEI and GSH was 1: 1, 1: 2, 1: 3, 1: 4. When the GST-eGFP fusion protein was introduced into the cell in the same manner as in Example 4, the conjugate using PEI having an average molecular weight of 600 showed higher ability to introduce the fusion protein. Okay (Figure 7).

  From this, the density and hydrophobicity in which about three glutathione bonds to the surface of PEI having an average molecular weight of 600 are compared with those of PEI having an average molecular weight of 1800, and the binding force between PEI-GSH and GST and the cell surface. It is thought that it contributes to the bond strength.

Synthesis of modified PEI (1) Synthesis of GX-1 Polyethyleneimine having an average molecular weight of 600 (Nippon Shokubai Co., Ltd., Epomin (trademark) SP-006) is dissolved in tetrahydrofuran (THF), and hexyl chloride is added to react. I let you. The obtained reaction solution was concentrated with an evaporator to obtain a target compound, which was designated as GX-1.
(2) Synthesis of GX-4 5 g of polyethyleneimine having an average molecular weight of 600 was dissolved in 15.3 g of tetrahydrofuran (THF), and 1.75 g of 1,3-butadiene monoepoxide (manufactured by Tokyo Chemical Industry Co., Ltd.) was added. The reaction was carried out at 5 ° C. for 5 hours. The obtained reaction solution was concentrated with an evaporator to obtain 6.8 g of the objective compound, which was designated as GX-4.
(3) Synthesis of GX-5 Polyethyleneimine having an average molecular weight of 600 was dissolved in tetrahydrofuran (THF) and reacted by adding styrene oxide. The obtained reaction solution was concentrated with an evaporator to obtain a target compound, which was designated as GX-5.

Intracellular introduction of fusion protein by modified PEI-GSH conjugate (1) Synthesis of modified PEI-GSH conjugate GX-4: γ-thiobutyrolactone: aldothiol synthesized in Example 7 = 1: 3: 3.1 The modified PEI-GSH conjugate was synthesized in the following molar ratio:

GX-4 0.2g (0.3 * 10 < ^-3 > mol) was added to the 30 ml eggplant type flask which put tetrahydrofuran (THF) 5ml. Under agitation, 0.24 g (1.1 × 10 ⁻³ mol) of aldrithiol and 0.1 g (1 × 10 ⁻³ mol) of γ-thiobutyrolactone were added and reacted at 50 ° C. for 1 hour. After THF was removed using an evaporator, 2 ml of ultrapure water was added to the reaction product and dissolved at 50 ° C. Since two-layer separation occurred after a while at room temperature, centrifugation was performed at 12,000 rpm for 5 minutes, and 500 μl of the lower layer portion was recovered. GX-4-GSH3 obtained by adding 74 mg of reduced glutathione (GSH) (0.23 × 10 ⁻³ mol, × 0.9 mol of γ-thiobutyrolactone) and reacting three GSHs with one GX-4 (150 mM at a concentration of GX-4) was synthesized and used in the following experiments.

Further, GX-1, also modified the GX-5 PEI: mixing ratio of GSH (coupling ratio) is 1: such that 2, GX-1 case of GX-1 0.2g (0.3 × 10 - ³ mol), 0.14 g (0.63 × 10 ⁻³ mol) of aldolthiol, 0.063 g (0.6 × 10 ⁻³ mol) of γ-thiobutyrolactone and 42 mg (0.14 × 10 ⁻³ mol) of GSH. ) In the case of GX-5, GX-5 0.2 g (0.3 × 10 ⁻³ mol), aldrithiol 0.14 g (0.63 × 10 ⁻³ mol), γ-thiobutyrolactone 0. GX-1-GSH2 conjugate and GX-5-GSH2 conjugate were prepared in the same manner as described above at a ratio of 063 g (0.6 × 10 ⁻³ mol) and GSH 16.6 mg (0.054 × 10 ⁻³ mol). Synthesized.

(2) Cell introduction of GST-eGFP using modified PEI-GSH In order to compare the cell introduction ability of GST fusion protein by PEI600 (unmodified) -GSH3 and GX-4-GSH3 synthesized in (1) above, A cell visualization experiment using GST-eGFP was performed.

The mouse fibroblast cell line Balb / c 3T3 A31H clone was spread on a 12-well plate containing a φ18 mm cover glass under the condition of 5 × 10 ⁴ cells / well. After culturing for 12 hours, the medium was replaced with 500 μl of Opti-MEM. GST-eGFP and PEI600-GSH3 or GX-4-GSH3 were mixed at a ratio of 1: 1 at a final concentration of 500 nM in the medium, reacted at room temperature for 5 minutes, and then added to the medium. The cells were cultured at 37 ° C. for 2 hours, and then cultured in Opti-MEM supplemented with 20 mM DTT for 10 minutes to remove proteins remaining on the cell surface. After washing with PBS, intracellular fluorescence localization was observed using a confocal laser microscope.

The state of the cells into which GST-eGFP has been introduced is shown in FIG.
When GST-eGFP was added alone, eGFP fluorescence was not observed in the cells (A in FIG. 8). When GST-eGFP was introduced using PEI600-GSH3, fluorescence on the grains was observed in the cells (B in FIG. 8). This seems to indicate that the protein taken up by endocytosis remains in the endosome. On the other hand, when GX4-GSH3 was used, in addition to cells exhibiting granular fluorescence, cells in which eGFP fluorescence spread uniformly in the cytoplasm could be confirmed (C in FIG. 8). The frequency of appearance was about 10% of the total cells.

  In addition, for GX-1-GSH2 conjugate and GX-5-GSH2 conjugate, a GST-eGFP-introduced cell visualization experiment was performed in the same manner as described above.

  FIG. 9 shows the results of a visualization experiment of GST-eGFP-introduced cells of GX-1-GSH2 conjugate, GX-4-GSH3 conjugate, and GX-5-GSH2 conjugate. The GX-4-GSH3 conjugate showed a particularly excellent introduction ability (a few percent of cells in which release into the cytoplasm is noticeable can be detected).

  Next, propidium iodide (PI) staining experiments were performed in order to determine whether the cells into which GST-eGFP had been introduced were viable or dead.

  The cells after introduction of GST-eGFP were cultured in a medium containing 1 μg / ml PI for 20 minutes, washed with PBS, and then observed using a confocal laser microscope. As a control experiment, PI staining of fixed cells was performed. The cells after introduction of GST-eGFP were reacted with a 4% paraformaldehyde solution for 30 minutes, and permeabilized in PBS containing 1% Triton X-100 for 10 minutes. The reaction was performed in a medium containing 1 μg / ml PI for 20 minutes, washed with PBS, and then observed using a confocal laser microscope (AD in FIG. 10).

  When PI was added to the observed cells, PI uptake could not be observed in both cells in which the fluorescence of eGFP was observed in a granular manner and cells in which the fluorescence was spread uniformly (B in FIG. 10). On the other hand, PI uptake could be observed in the cells subjected to the immobilization treatment (D in FIG. 10).

  Based on the above results, the GX-4-GSH3 conjugate has the ability to positively release the GST fusion protein taken up by endocytosis into the cytoplasm, and is a useful carrier that does not damage cells. There was found.

Confirmation of MAPK cascade reaction in vitro by GST-CA-MEK1 Example except that plasmid DNA (pFC-MEK1, Stratagene) encoding constitutively active MEK1 (CA-MEK1) is used In the same manner as in Example 1, a GST-CA-MEK1 fusion protein was prepared. In CA-MEK1, the Leu33 to Ala52 residues are deleted, Ser217 is replaced with Glu217, and Ser221 is replaced with Glu221.

  Both or one of CA-MEK1 and ERK1 expressed as a GST fusion protein is mixed with MBP and ATP in a test tube. When ERK1 is activated by CA-MEK1, MBP is subsequently phosphorylated. Then, phosphorylated MBP was detected by Western blotting using anti-phospho MBP antibody (anti-p-MBP antibody). As a result, phosphorylated MBP was observed only in the sample in which both CA-MEK1 and ERK1 were mixed (FIG. 11).

Confirmation of MAPK cascade reaction at the cellular level by GST-CA-MEK1 Using 300 nM of PEI600-GSH3 conjugate to the cell HLR-Elk1 cell / Stratagene, which can evaluate the activation level of ERK in cells by luciferase activity 300 nM GST-CA-MEK1 fusion protein was introduced. GST-CA-MEK1 introduced into the cell activates endogenous ERK, and subsequently activates GAL4BD-Elk1 expressed in the cell in advance. Activated GAL4BD-Elk1 induces expression of a reporter gene (luciferase) integrated on the genome. As a result, the introduction of GST-CA-MEK1 using PEI600-GSH3 could be confirmed by quantifying luciferase activity. The increase in luciferase activity obtained here was confirmed to be about the same as that in EGF stimulation, but such increase in activity was confirmed in cells that had not been treated or only GST was introduced with PEI600-GSH3. Not (Figure 12).

  The conjugate of the present invention easily binds to the GST fusion protein and can introduce the target protein into the cells by a simple operation. For this reason, GST fusion protein which is widely used all over the world can be further utilized as a research asset. By using the conjugate of the present invention, the glutathione S transferase fusion protein can be used in living cells, and therefore various applications are expected to be created. In addition, it is expected as a research reagent that can greatly contribute to research fields such as post-translational modification of proteins.

The synthesis scheme of the conjugate of the present invention is shown. A mode that a PEI-GSH-GST fusion protein is introduce | transduced into a cell is shown typically. Intracellular introduction of PEI-GSH-GST by Western blot is shown. A: Optimization of the mixing ratio of GST and PEI600-GSH conjugate is shown. B: shows the time course of intracellular introduction of GST and PEI600-GSH conjugate (mixing ratio (molar ratio) 1:10). C: shows concentration-dependent intracellular introduction of GST and PEI600-GSH conjugate (mixing ratio (molar ratio) 1:10). The uptake | capture into the cell of GST-eGFP fusion protein by a confocal laser scanning microscope is shown. The cells observed with a Cre / LoxP system and a fluorescence microscope are shown. A: A reporter gene for confirming the expression of GST-Cre recombinase activity in cells. B: shows a CHO cell (stable expression strain) into which the A reporter gene has been incorporated. C: The cell 2 days after adding GST-Cre recombinase + PEI600-GSH to the cell of B is shown. The intracellular introduction | transduction of GST-eGFP by PEI600-GSH is shown. The intracellular introduction | transduction of GST-eGFP by PEI1800-GSH and PEI600-GSH is shown. Intracellular introduction of GST-eGFP fusion protein is shown. A: The case where GST-eGFP is added alone is shown. B: The case where GST-eGFP is introduced using PEI600-GSH3 is shown. C: The case where GST-eGFP is introduced using GX-4-GSH3 is shown. Intracellular introduction of GST-eGFP fusion protein using modified PEI-GSH is shown. The PI staining of GST-eGFP-introduced cells is shown. A: eGFP fluorescence in living cells. B: PI staining in live cells. C: eGFP fluorescence in fixed cells. D: PI staining in immobilized cells. Figure 3 shows confirmation of MAPK cascade reaction in vitro. The introduction of GST-CA-MEK1 into HLR-ELK cells is shown.

Claims (23)

A conjugate in which polyethyleneimine or a chemically modified derivative thereof and glutathione are bound at a molar ratio of 1: 2 to 1: 3 . The conjugate according to claim 1, wherein polyethyleneimine or a chemically modified derivative thereof and glutathione are bound at a molar ratio of 1: 3. The conjugate according to claim 1 or 2 , wherein the polyethyleneimine has a number average molecular weight in the range of 100 to 100,000. The conjugate according to any one of claims 1 to 3 , wherein the polyethyleneimine has a branched structure. The conjugate according to any one of claims 1 to 4 , wherein the chemically modified derivative is an N-alkyl derivative, N-acyl derivative, N-sulfonyl derivative or Schiff base derivative of polyethyleneimine. The conjugate according to any one of claims 1 to 5 , wherein the glutathione is a reduced glutathione. The conjugate according to any one of claims 1 to 6 , wherein polyethyleneimine and glutathione are bonded via a disulfide bond. A complex of the conjugate according to any one of claims 1 to 7 and a glutathione S-transferase fusion protein. 9. The complex of claim 8 , wherein the conjugate and glutathione S transferase fusion protein are bound at a molar ratio of 1:10 to 100: 1. Polyethyleneimine or its chemically modified derivative and 2,2-dipyridyl disulfide are reacted with the amino group of polyethyleneimine or its chemically modified derivative in the presence of a reagent that exposes the thiol group and reduced to the resulting product. A method for producing a conjugate according to any one of claims 1 to 7 , comprising a step of contacting the type glutathione. The method according to claim 10 , wherein the reagent is γ-thiobutyrolactone, methyl 4-mercaptoalkaneimidate, 2-iminothiolane or N-acetylhomocysteine thiolactone. The method according to claim 10 or 11 , wherein the chemically modified derivative is an N-alkyl derivative, N-acyl derivative, N-sulfonyl derivative or Schiff base derivative of polyethyleneimine. A complex according to claim 8 or 9 , comprising mixing the conjugate according to any one of claims 1 to 7 with a glutathione S-transferase fusion protein to form a complex thereof. Method. 14. The method of claim 13 , comprising mixing the conjugate and glutathione S transferase fusion protein in a molar ratio of 1: 1 to 100: 1. Glutathione S-transferase fused to a gene encoding the gene and the target protein coding to produce a fusion gene, further comprising the step of obtaining a glutathione S-transferase fusion protein The fusion gene was expressed in E. coli, according to claim 13 or 14 The method described in 1. A method for introducing a protein into a cell, comprising transporting the target protein into a cell in vitro using the conjugate according to any one of claims 1 to 7 . 17. The method of claim 16 , comprising mixing the conjugate and glutathione S transferase fusion protein to form a complex thereof. The method according to claim 17 , wherein the mixing ratio of the conjugate and glutathione S-transferase fusion protein is 1: 1 to 100: 1 in molar ratio. A method for introducing a protein of interest into a cell, comprising transporting the protein into a cell in vitro using the complex according to claim 8 or 9 . A protein intracellular introduction agent comprising the conjugate according to any one of claims 1 to 7 . A protein intracellular introduction agent comprising the complex according to claim 8 or 9 . A protein intracellular introduction kit comprising the protein intracellular introduction agent according to claim 20 . A protein intracellular introduction kit comprising the protein intracellular introduction agent according to claim 21 .

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