KR20200113764A - Method for preparing polyglutamate-TAT-Cre fusion protein - Google Patents

Abstract

The present invention provides: a method of producing polyglutamate-TAT-Cre fusion proteins, which comprises the steps of: (a) inserting a polyglutamate sequence consisting of an amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene; (b) expressing the polyglutamate-TAT-Cre gene obtained in the step (a) to obtain a polyglutamate-TAT-Cre recombinant protein; and (c) purifying the polyglutamate-TAT-Cre recombinant protein obtained in the step (b); and a method of producing a TAT-Cre protein, which comprises the steps of: making the polyglutamate-TAT-Cre recombinant protein to react with TEV protease to obtain a product; and purifying the obtained product so as to obtain a TAT-Cre protein. The method of producing polyglutamate-TAT-Cre fusion proteins according to the present invention is a method of producing effective proteins used in treatment and research, and thus can be usefully used in the medical and pharmaceutical fields.

Description

Method for preparing polyglutamate-TAT-Cre fusion protein {Method for preparing polyglutamate-TAT-Cre fusion protein}

The present invention relates to a method for preparing a polyglutamate-TAT-Cre fusion protein, and more particularly, a polyglutamate-TAT-Cre expressed by inserting a polyglutamate sequence consisting of amino acid SEQ ID NO: 1 into a gene encoding TAT-Cre. It relates to a fusion protein.

Cell penetrating peptides (CPPs), also called protein transduction domains (PTDs) or Trojan peptides, enable efficient intracellular delivery of a variety of proteins with rare toxicity. CPP is an oligopeptide of 5 to 30 amino acid residues in length, such as HIV-1 derived TAT _47-57 (YGRKKRRQRRR), polyarginine 9 (RRRRRRRRR) and _{phenerratin 43-57} (RQIKIWFQNRRMKWKK). TAT, a trans-activator of transcription, is a basic peptide (arginine and lysine) and acts as a PTD. TAT _47-57 , an amphiphilic and strong cation, is internalized into cells through receptor-dependent lipid raft-dependent macropinocytosis. Cargo delivered to the cytoplasm by TAT PTD is translocated from the SV-40 giant T antigen to the nucleus due to the nuclear localization sequence (NLS).

Various proteins containing the cationic CPP tag sequence were produced in E. coli. Nevertheless, in some cases, insoluble protein aggregates known as inclusion bodies in the cytoplasm or between the inner and outer membranes of cells when the foreign protein expressed in E. coli constitutes more than 2% of the total cellular protein. Create In order to produce a functionally active protein, it is necessary to purify the inclusion body, and the inclusion body is separated, solubilization by denaturation with a chaotropic agent, refolding, and purification of soluble proteins from refolding reactions. A cumbersome process is required to restore the native-like conformation. A certain amount of insoluble protein refolds correctly, and the exact degree of refolding depends on the nature of the protein sequence as well as the choice for the refolding procedure. In general, a denaturant, a surfactant, or a high concentration (2 to 8 M) of urea or guanidine hydrochloride is added to release the aggregated protein by restoring its shape.

Cre recombinase has been widely used to induce recombination of site-specific genes or to regulate the expression of a target gene in vitro or in vivo . The two loxP sites, each 34 bp of Cre recognition sites, flank a target site called "floxed allele". Cre-mediated recombination results in fusion to the two flanking loxP sites, releasing these DNA segments. Also, a floxed reporter gene such as β-galactosidase or an intracellular fluorescent protein visually indicates the effect of Cre. Cre with TAT fusion has been used to enhance the penetration of Cre into cells.

Prior literature [Q. According to Lin, et al., BMC Biotechnol 4 (2004) 25], although the amount of expressed TAT-Cre was less than that of unfused Cre, the solubility of unfused Cre was relatively higher than that of TAT-Cre. It was high. Judging from these results, it was inferred that the strong cationic TAT could be the cause of the low solubility of TAT-Cre in the E. coli cytoplasm. Therefore, there is a need to develop a technology capable of improving the low solubility of TAT-Cre.

Korean Patent Application Publication No. 10-2018-0006402 (2018.01.17) Korean Patent Application Publication No. 10-2016-0013730 (2016.02.05)

Lilie, H., et al., Biol. Chem. 2013; 94(8):995-1004 Biochemistry. 2010; 49(36):7854-7866

The inventors of the present invention confirmed whether the addition of a negatively charged polyglutamate sequence (EEEEEGSEEEEEEE, SEQ ID NO: 1) can improve the solubility of TAT-Cre and the production yield in E. coli. Once the polyglutamate-attached TAT-Cre described in SEQ ID NO: 1 was purified and treated with TEV protease, TAT-Cre having high purity and activity was obtained, and the yield was found to be higher compared to the expression of TAT-Cre alone. I did.

Accordingly, an object of the present invention is to provide a method for producing a polyglutamate-TAT-Cre fusion protein.

According to one aspect of the present invention, (a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene; (b) expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein; And (c) there is provided a method for producing a polyglutamate-TAT-Cre fusion protein comprising the step of purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b).

In one embodiment, the polyglutamate-TAT-Cre gene of step (a) may further include any one of a His tag and a GST tag, and may further include a TEV protease recognition site. In addition, the polyglutamate-TAT-Cre gene of step (a) is at least one selected from the group consisting of glycine (G), methionine (M) and alanine (A) at the N-terminus of the TAT peptide. It may further comprise an amino acid residue.

In one embodiment, in step (b), the expression may be induced with 0.05 to 5 mM IPTG, and the IPTG may be induced in the host cell for 1 to 5 hours. In addition, the expression may be performed using E. coli as a host cell.

According to another aspect of the present invention, (a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene; (b) expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein; (c) purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b); (d) reacting the polyglutamate-TAT-Cre recombinant protein obtained in step (c) with TEV protease to obtain a product; And (e) there is provided a method for producing a TAT-Cre protein comprising the step of purifying the product obtained in step (d) to obtain a TAT-Cre protein.

In one embodiment, the reaction in step (d) may be carried out at 25 to 35 °C, and may be carried out at pH 7.0 to 9.0.

According to another aspect of the present invention, there is provided a polyglutamate-TAT-Cre fusion protein prepared by the above production method.

In one embodiment, the protein may be 46 kDa.

According to another aspect of the present invention, there is provided a TAT-Cre protein prepared by the above production method.

In one embodiment, the protein may be 42 kDa.

According to another aspect of the present invention, the polyglutamate-TAT-Cre fusion protein; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is 1 selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast substances, drugs and chemicals. More than a species, a cargo delivery system is provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for cell penetration comprising the polyglutamate-TAT-Cre fusion protein.

According to another aspect of the present invention, the TAT-Cre protein; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is 1 selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast substances, drugs and chemicals. More than a species, a cargo delivery system is provided.

According to another aspect of the present invention, there is provided a pharmaceutical composition for cell penetration comprising the TAT-Cre protein.

According to the present invention, it was found that the polyglutamate-TAT-Cre fusion protein inserted with the polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 improves the solubility and yield of the recombinant TAT-Cre protein. In addition, it was confirmed that the production of the target protein was increased by increasing the growth of cells expressing polyglutamate-TAT-Cre.

Therefore, the method for preparing a polyglutamate-TAT-Cre fusion protein of the present invention can be usefully used in medicine and pharmaceutical fields as a method for preparing an effective protein used for treatment and research.

1 shows the construction and amino acid sequence of recombinant H6-E12-TAT-Cre. 1A is a schematic diagram showing the construction of a recombinant H6-E12-TAT-Cre, and FIG. 1B is a polyglutamate (E12) and TAT-NLS-Cre cloned into pETM11 vector carrying a His tag (H6) and a TEV protease cleavage site. Is the amino acid sequence of.
Figure 2 shows the expression of cellular proteins for H6-TAT-Cre or H6-E12-TAT-Cre after IPTG induction. Fig. 2A is an SDS-polyacrylamide gel stained with Coomassie blue of His-tagged TAT-Cre with or without E12 in total cellular protein. This staining (in 12% gel after SDS-PAGE) was performed on the soluble and insoluble fractions in E. coli transformed with H6-TAT-Cre (42 kDa) or H6-E12-TAT-Cre (46 kDa) without E12. Total cellular protein was visualized. 2B is a Western blotting result of analyzing TAT-Cre tagged with His tag with or without E12. His tagged proteins were detected by anti-His tagged antibodies at the level of 42 kDa (H6-TAT-Cre) or 46 kDa (H6-E12-TAT-Cre). GAPDH (35 kDa) was used as a loading control.
3 shows the growth of the E. coli strain expressing the recombinant protein and purification of the recombinant TAT-Cre protein. 3A is a graph showing the growth of cells transformed with TAT-Cre or E12-TAT-Cre. Growth was evaluated every hour by measuring the absorbance at 600 nm until the stationary phase. IPTG induction expressing TAT-Cre was started after incubation at 37° C. for 3 hours at the exponential growth stage of E. coli. 3B is a result showing the purification of TAT-Cre by Ni-affinity chromatography. Lysates of IPTG-induced cells were subjected to Coomassie blue staining. Cell lysates were obtained from H6-E12-TAT-Cre plasmid-transformed E. coli by sonication. The first fluid (flow-through, 1st FT) did not contain H6-E12-TAT-Cre because the His-tagged protein binds to the Ni-column and other components of the cell lysate pass through the column. The column was washed with a buffer containing 30 mM imidazole, and H6-E12-TAT-Cre (46 kDa) was eluted with an elution buffer. After treatment with TEV protease, the eluate was passed through the column again to separate His tagged H6-E12 attached to the column, and TAT-Cre contained in the second emulsion (flow-through, 2nd FT) was obtained. M represents a molecular size marker.
Fig. 4 shows a cell-free assay for Cre recombination activity of recombinant TAT-Cre and cytotoxicity of TAT-Cre on 293FT cells. 4A is a schematic diagram schematically showing the analysis of TAT-Cre activity in a cell-free system. The pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE was digested with HindIII to linearize it, and the recombinant enzyme activity of the purified TAT-Cre was evaluated. Figure 4B is a gel photograph showing a cell-free analysis of the recombinant TAT-Cre activity. HindIII-linearized plasmids were reacted with various concentrations of TAT-Cre. The mixture was reacted at 37° C. for 1 hour and heated at 70° C. for 10 minutes to inactivate TAT-Cre. Recombination of plasmid DNA was analyzed by agarose gel electrophoresis. M represents a 1 kb ladder marker. 4C is a graph showing the survival rate of mammalian 293FT cells exposed to TAT-Cre. Cells were treated with various concentrations of TAT-Cre for 24 hours. After reacting, cell viability was measured by CCK-8 analysis. Results are expressed as a percentage of the control group treated with a solvent. Data are expressed as mean ± standard deviation. * Indicates that p<0.05 compared to the control group (n=3) treated with 50% glycerol (vehicle).
5 is a fluorescence microscope image showing TAT-Cre conversion efficiency in 293FT cells. 293FT cells were transfected with pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE through Lipofectamine 3000 for 24 hours. After that, the TAT-Cre protein was administered at various concentrations, and the cells were reacted at 37° C. and 5% CO ₂ for 24 or 48 hours. The reacted cells were imaged using a fluorescence microscope.

In the present invention, (a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene; (b) the polyglutamate obtained in step (a) -Expressing the TAT-Cre gene to obtain a polyglutamate-TAT-Cre recombinant protein; And (c) it provides a method for producing a polyglutamate-TAT-Cre fusion protein comprising the step of purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b).

The manufacturing method of the present invention includes a step of obtaining a polyglutamate-TAT-Cre gene by inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre (ie, step (a)). Step (a) is a step of constructing a plasmid expressing a polyglutamate-TAT-Cre gene by inserting a polyglutamate (E12) sequence into a plasmid encoding TAT-Cre.

TAT-Cre plays a role in helping the passage of cell membranes when manipulating DNA sequences in vitro or in vivo . However, there is a problem in that the yield of recombinant TAT-Cre is low because the TAT sequence rich in arginine with a strong positive charge forms an inclusion body. To solve this problem, a negatively charged polyglutamate (E12) sequence can be inserted in the vicinity of the positively charged TAT peptide to neutralize the TAT peptide.

In addition, the polyglutamate-TAT-Cre gene may further include any one of a His tag and a GST tag, and preferably may further include a His tag. Since the His-tagged protein binds to the Ni-column, the protein can be obtained using a Ni-affinity chromatography method.

In addition, the polyglutamate-TAT-Cre gene may further include a TEV protease recognition site. TEV (Tobacco etch virus) protease is an intranuclear proteolytic enzyme produced by TEV of 27 kDa. TEV protease can be produced by putting the recognition sequence of the TEV protease at the C-terminus or N-terminus of the protein to be produced and fusing His tag or GST tag to produce a recombinant protein. The recombinant protein thus produced can be treated with TEV protease to remove the fused tag after separating and purifying the protein with high purity using the affinity of the tag.

In addition, the polyglutamate-TAT-Cre gene is glycine (G), methionine (M), and alanine at the N-terminus of the TAT peptide to ensure flexibility and prevent another possible steric hindrance due to TEV cleavage. It may further include one or more amino acid residues selected from the group consisting of alanine, A) (see FIG. 1B).

The manufacturing method of the present invention includes a step of expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein [ie, step (b)]. Step (b) is a step of inducing the expression of the polyglutamate-TAT-Cre recombinant protein in E. coli using IPTG.

In step (b), the expression is IPTG 0.05 to 5 mM, preferably 0.1 to 2 mM, most preferably 0.5 mM in the host cell for 1 to 5 hours, preferably for 2 to 4 hours, most preferably May be induced for 3 hours. In addition, the IPTG may be induced at 35 to 40 °C, preferably at 37 °C. The expressed protein can be obtained from a host cell, and specifically, after lysis by adding a lysis buffer to the host cell, it can be obtained as a supernatant or pellet by a method such as centrifugation.

The production method of the present invention includes the step of purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b) [ie, step (c)]. Step (c) is a step of purifying the polyglutamate-TAT-Cre recombinant protein by Ni-affinity chromatography. The polyglutamate-TAT-Cre recombinant protein obtained in step (b) was loaded on a Ni-column, washed with a buffer containing 30 mM imidazole, and then purified by Ni-affinity chromatography eluting with an elution buffer. have.

The polyglutamate-TAT-Cre fusion protein may be prepared in E. coli.

Host cells capable of stably and continuously cloning and expressing the plasmid encoding the polyglutamate-TAT-Cre gene of the present invention may preferably be E. coli, such as E. coli BL21 (DE3), E. coli C43 (DE3), E. coli JM109, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776 and E. coli W3110.

The method of transforming the plasmid encoding the gene of the present invention into a host cell is a heat shock method, a CaCl ₂ method [Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973)], one method [Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114 (1973); And Hanahan, D., J. Mol. Biol., 166:557-580 (1983)] and electroporation methods [Dower, WJ et al., Nucleic. Acids Res., 16:6127-6145 (1988)] and the like, and commercially available transformation kits can be used.

In addition, the present invention includes the steps of (a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene; (b) expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein; (c) purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b); (d) reacting the polyglutamate-TAT-Cre recombinant protein obtained in step (c) with TEV protease to obtain a product; And (e) it provides a method for producing a TAT-Cre protein comprising the step of obtaining a TAT-Cre protein by purifying the product obtained in step (d).

In the above manufacturing method, steps (a) to (c) are as described in the manufacturing method of the polyglutamate-TAT-Cre fusion protein.

The present invention includes the step of obtaining a product by reacting the polyglutamate-TAT-Cre recombinant protein obtained in step (c) with TEV protease [ie, (d)]. Step (d) is a step of separating polyglutamate from the polyglutamate-TAT-Cre fusion protein. Since the polyglutamate-TAT-Cre fusion protein has a TEV protease recognition site, it can be reacted with TEV protease to easily remove polyglutamate.

Here, the reaction is 25 to 35 °C, preferably 27 to 33 °C, most preferably at 30 °C, pH 7.0 to pH 9.0, preferably pH 7.5 to pH 8.5, most preferably pH 8.0 to 0.5 to It may be performed for 3 hours, preferably for 1 to 2 hours, and most preferably for 1 hour.

The production method of the present invention includes a step of purifying the product obtained in step (d) to obtain a TAT-Cre protein [ie, (e)]. Step (e) is a step of purifying the TAT-Cre protein from which the polyglutamate is separated by Ni-affinity chromatography. The TEV protease-treated product obtained in step (d) was reloaded on a Ni-column and subjected to secondary purification by Ni-affinity chromatography, so that the His-tagged polyglutamate remains on the column, and the TAT-Cre protein remains in an emulsion (flow -through), you can easily obtain TAT-Cre protein.

In addition, the present invention provides a polyglutamate-TAT-Cre fusion protein prepared by the above production method.

The polyglutamate-TAT-Cre fusion protein is a protein having a size of 46 kDa, and polyglutamate is inserted to increase the total protein amount of the target protein, as well as increase the amount of soluble protein and decrease the amount of insoluble protein. Confirmed (see Table 1). In addition, it was confirmed that the growth rate was increased in the host cell transformed with the polyglutamate-TAT-Cre plasmid compared to the host cell transformed with the TAT-Cre plasmid. Therefore, the method for preparing a polyglutamate-TAT-Cre fusion protein of the present invention can be usefully used in medicine and pharmaceutical fields as a method for preparing an effective protein used for treatment and research.

In addition, the present invention provides a TAT-Cre protein prepared by the above production method.

The TAT-Cre protein was a protein having a size of 42 kDa, and it was confirmed that the yield was increased by 1.5 times or more, and the activity of the TAT-Cre protein was maintained in cell-free systems and mammalian cells. Accordingly, it was confirmed that inserting the polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 is a novel method for improving the expression and purification of the target protein without affecting the activity of the TAT-Cre protein.

In addition, the present invention is the polyglutamate-TAT-Cre fusion protein; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is 1 selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast substances, drugs and chemicals. It provides a cargo delivery system that is more than a species.

The cargo includes all substances that can move into the cell by binding with the cell-permeable peptide, for example, all substances desired to increase the cell permeation efficiency, specifically the effective substance of the drug, more specifically proteins, nucleic acids, peptides, Examples include, but are not limited to, sugars, nanoparticles, biological agents, viruses, contrast media, or other chemical substances, for example minerals and glucose.

The drug is a broad concept including substances for alleviating, preventing, treating or diagnosing a disease, upper body or specific symptoms.

In addition, the present invention provides a pharmaceutical composition for cell penetration comprising the polyglutamate-TAT-Cre fusion protein.

The polyglutamate-TAT-Cre fusion protein can be used pharmaceutically as a delivery vehicle that efficiently permeates the active ingredient, Cargo, into cells. Through this, the efficacy of the active ingredient can be increased, and as a result, the dosage can be lowered, thereby minimizing side effects caused by drug administration and increasing treatment efficiency.

The pharmaceutical composition may include a concentration of 1 to 1,000 μg/ml, preferably a concentration of 1 to 700 μg/ml, but is not limited thereto, and the body weight, age, sex, health status, diet, administration time of the subject to be administered , It may vary depending on the method of administration, the rate of excretion and the severity of the disease. In addition, the pharmaceutical composition may be administered orally, rectal, transdermal, intravenous, intramuscular, intraperitoneal, intramedullary, intrathecal or subcutaneous according to a desired method.

In one embodiment, the pharmaceutical composition may further include one or more pharmaceutically acceptable carriers selected from the group consisting of fillers, extenders, binders, wetting agents, disintegrants, surfactants, diluents and excipients.

Formulations for oral administration may be tablets, pills, soft or hard capsules, granules, powders, liquids, or emulsions, but are not limited thereto. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but are not limited thereto.

In addition, the pharmaceutical composition may contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavors or sweeteners, if necessary. The pharmaceutical composition of the present invention can be prepared by conventional methods in the art.

In addition, the present invention is the TAT-Cre protein; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is 1 selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast substances, drugs and chemicals. It provides a cargo delivery system that is more than a species.

In addition, the present invention provides a pharmaceutical composition for cell penetration containing the TAT-Cre protein.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Materials and methods

(1) Preparation of prokaryotic plasmid

pETM11 contains a His tag (H6) and a T7 promoter followed by a TEV protease recognition site (TEV site). An oligonucleotide encoding E12 (EEEEEGSEEEEEEE, SEQ ID NO: 1) was inserted between the TEV site of H6 and pETM11 through in vitro mutagenesis based on PCR. DNA fragments encoded with TAT peptide (GRKKRRQRRRPPAGTSVSL), fragments encoded with NLS (KKKRKV) and fragments encoded with Cre were assembled at the C-terminus of the TEV site using PCR. The exact structure of the plasmid was confirmed through sequencing.

(2) Expression and purification of recombinant protein

The Cre expression plasmid, H6-TAT-Cre or H6-E12-TAT-Cre was transfected into BL21 (DE3) cells and then incubated overnight in 1 L of Luria-Bertani (LB) medium supplemented with 40 µg/ml kanamycin. I did. Expression of the recombinant protein was induced at 37° C. for 3 hours by treatment with 0.5 mM isopropyl β- _D -1-thiogalactopyranoside (IPTG). Cell growth was measured every hour for 10 hours at an optical density (absorbance) of 600 nm (OD ₆₀₀ ). Cells were collected by centrifugation, and a lysis buffer [5 mM imidazole, 500 mM NaCl, 5% glycerol, 4 mM β-mercaptoethanol, and 20 mM Tris-HCl, pH 8.0] was added to the cells to be ultrasonicated. Processed. The cell lysate was centrifuged at 4° C. for 20 minutes at 15,000×g to obtain a supernatant and a pellet (containing inclusion bodies).

Each His-tagged protein in the supernatant was purified through Ni-NTA Agarose Beads (QIAGEN), and a washing buffer (30 mM imidazole, 500 mM NaCl, 5% glycerol, 4 mM β-mercaptoethanol) , 0.05% Tween 20 and 20 mM Tris-HCl, pH 8.0]. His tagged TAT-Cre and E12-TAT-Cre were used in elution buffer (250 mM imidazole, 500 mM NaCl, 5% glycerol, 4 mM β-mercaptoethanol, 0.05% Tween 20 and 20 mM Tris-HCl. , pH 8.0].

After that, the eluate was first reacted with TEV protease at pH 8.0 at 30° C. for 1 hour, precipitated with ammonium sulfate, and then concentrated with a lysis buffer. The reaction mixture was again subjected to Ni-affinity chromatography. The eluted second emulsion (2nd flow-through) was dialyzed overnight at 4° C. in a dialysis buffer [500 mM NaCl, 50% glycerol and 20 mM Tris-HCl, pH 7.5] in SnakeSkin™ Dialysis Tubing (Thermo Scientific). Finally, the TAT-Cre protein was sterilized with a Costar Spin-X 8160 Centrifuge tube filter (Thermo Scientific).

(3) Coomassie blue staining and Western blot analysis

After centrifugation at 15,000 × g for 10 minutes, unpurified supernatant and pellet (containing inclusion bodies) were obtained from cells transformed with H6-TAT-Cre or H6-E12-TAT-Cre plasmid. The pellet was washed with 20 mM Tris-HCl (pH 7.5), heated to 85° C., completely denatured with 1% SDS solution, and sonicated until completely solubilized. The solution of the SDS-treated pellet was combined with the supernatant and expressed as "total cell protein". The protein level was measured using a BCA protein analysis reagent (Thermo Scientific).

30 μg of protein was separated on a 12% gel by SDS-PAGE and stained with Coomassie blue or transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 3% bovine serum albumin (BSA) for 1 hour and reacted with His probe-HRP [1:5000 dilution with 3% BSA, Thermo Scientific] for 1 hour. Mouse anti-GAPDH antibody [1:5000 dilution with Tris-buffered saline (TBST) containing 0.1% Tween 20, Thermo Scientific] was reacted with the membrane for 1 hour, and then HRP-conjugated mouse antibody [with TBST] 1: 5000 dilution, Thermo Scientific] was reacted for 1 hour. The blot was developed using Super Signal West Pico Chemiluminescent Substrate (Pierce).

(4) In vitro analysis for Cre activity test

The pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE plasmid purchased from Addgene (USA) was digested with HindIII to linearize, and TAT-Cre of various concentrations and 50 μl of reaction buffer [33] at 37° C. for 1 hour mM NaCl, 50 mM Tris-HCl and 10 mM MgCl ₂ , pH 7.5] and 300 ng of linearized plasmid were added. The solution was heated at 70° C. for 10 minutes to inactivate Cre, and DNA fragments were separated from the protein by phenol extraction and EtOH precipitation. The separated DNA was analyzed by agarose gel electrophoresis.

(5) Cell viability evaluation

293FT cells were cultured at 37° C. and 5% CO ₂ in high-glucose DMEM supplemented with 10% FBS, 1% penicillin/streptomycin solution, and 1% non-essential amino acid solution. Cell viability was evaluated through Cell Counting Kit-8 (CCK-8) analysis. 293FT cells were plated at 10 ⁴ cells/well on a 96-well plate coated with 0.1% gelatin solution, and treated with TAT-Cre at various concentrations for 24 or 48 hours. Next, 10% of the CCK-8 colorimetric solution (relative to the total reaction amount) was added to each well, and further incubated for 4 hours in a humidified incubator. Then, the absorbance at 450 nm (OD ₄₅₀ ) was measured and normalized to OD ₆₅₀ .

(6) fluorescence imaging

10 ⁵ cells/well of 293FT cells were plated on a 12-well plate coated with 0.1% gelatin solution and incubated overnight to confirm 60-70% confluence. Cells were transfected with 300 ng of DsRed-floxed plasmid using Lipofectamine 3000 (Invitrogen). After 24 hours, pre-sterilization with serum-free medium at different doses (0, 1, 2, 4 and 6 μM) for different periods (0, 4, 6, 8, 12, 24 and 48 hours) One TAT-Cre was treated. Cell images were obtained at a wavelength of 480 nm or 580 nm using a fluorescence microscope (Olympus, CKX53).

2. Results

(1) Confirmation of improvement of soluble TAT-Cre protein expression in E. coli through addition of polyglutamate sequence

To confirm the solubility of the TAT-Cre protein, pETM11-TAT-NLS-Cre, a prokaryotic expression vector, was used as a control, and pETM11-E12-TAT-NLS-Cre was constructed according to the schematic diagram of FIG. 1A. In order to prevent steric hindrance by successive glutamate residues, glycine (G) and serine (S) were added in the E12 domain (see Fig. 1B). Several amino acid residues such as glycine (G), methionine (M) and alanine (A) were inserted into the N-terminal TAT tag to ensure flexibility and prevent another possible steric hindrance due to TEV cleavage ( 1B).

In the case of cells expressing E12-TAT-Cre protein, the amount of soluble protein significantly increased and the amount of insoluble protein decreased after IPTG induction compared to E. coli expressing the TAT-Cre protein. In addition, according to the purification results, the amount of soluble TAT-Cre (~ 7.9 mg/L) increased when the E12 domain was added compared to the amount of TAT-Cre (~ 4.9 mg/L) excluding E12 (Table 1). Reference).

Corresponding SDS-PAGE and Western blot data confirmed that the solubility of TAT-Cre was increased by E12 (see FIGS. 2A and 2B). The results show that the negatively charged E12 sequence of the protein successfully neutralizes the TAT sequence rich in positively charged amino acids, followed by enhancing the solubility of the protein. Moreover, it appears that neutralization of the TAT sequence under the same IPTG induction conditions leads to higher expression levels and improved solubility of the E12-TAT-Cre protein (see Table 1). Cells transformed with the TAT-Cre plasmid from which E12 was excluded were able to grow up to OD ₆₀₀ = about 8.8. In contrast, cells transformed with the E12-TAT-Cre plasmid were able to reach OD ₆₀₀ = about 9.6. These results suggest that the lower expression level of TAT-Cre excluding E12 may be due in part to inhibition of cell growth by positively charged TAT peptides (see FIG. 3A).

(2) Purification and activity measurement of TAT-Cre

His tagged E12-TAT-Cre protein (46 kDa) was purified through two consecutive Ni-affinity chromatography steps, and cleavage reaction by TEV protease was performed between the two steps (see Fig. 1A). Once the cell lysate was prepared, it was loaded onto a Ni-NTA affinity column, and the His-tagged protein was bound to the resin of the column. Thereafter, the His-tagged protein was eluted with a higher imidazole concentration. The eluate was treated with TEV protease, and the recognition/cleavage sequence located between E12 and TAT was cut. The obtained degradation products were His tag/E12 and TAT-Cre recombinant enzymes. The obtained product was loaded onto the added Ni-NTA column. The TAT-Cre recombinant enzyme did not bind to the Ni-NTA resin because it did not have a His tag. Therefore, the emulsion fraction was collected during the second Ni-NTA chromatography to recover the high-purity TAT-Cre recombinant enzyme (42 kDa). , His tag/E12 was retained in the column (see Figure 3B).

Through this purification method, high-purity TAT-Cre was obtained, and it was confirmed that the yield was significantly higher than that of the control group (4.9 mg/L) at 7.9 mg/L (see Table 1).

(3) Evaluation of the activity of recombinant TAT-Cre in cell-free system and 293FT cells

In order to confirm the activity of purified TAT-Cre, in vitro recombination assay was performed in a cell-free system. The pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE plasmid (8.5 kb) was linearized with HindIII (see Fig. 4A). Subsequently, the linearized vector was used as a substrate for the purified TAT-Cre recombinant enzyme. As a result, the floxed-DsRed gene was recombined in a dose-dependent manner by the TAT-Cre recombinase. Agarose gel electrophoresis produced a linearized pMSCV-loxP-eGFP-Puro-WPRE band (7788 bp, see Fig. 4B) as a recombinant product, whereas other reaction products of floxed-DsRed (712 bp) in a small circular shape were small. Because of its size, it was not visible in the gel. When 40 ng of TAT-Cre was reacted with 300 ng of plasmid, the LoxP-Cre recombination reaction was maximized.

Next, it was confirmed whether the recombinant TAT-Cre has functional activity in mammalian cells. Since DsRed is flanked by loxP in the pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE plasmid, active TAT-Cre removes DsRed and pMSCV-loxP-eGFP-Puro-WPRE through site-specific recombination. Can be created. 293FT cells were transfected with pMSCV-loxP-DsRed-loxP-eGFP-Puro-WPRE, and when recombinant enzyme treatment was not performed, the transfected cells expressed only red fluorescent protein (RFP) due to the proximal promoter. Started to do. In the absence of recombination, transcription was initiated upstream of DsRed, and because there was a stop codon immediately after the DsRFP open reading frame (ORF), the enhanced green fluorescent protein (eGFP) from the transcription protein before. Translation has been stopped. In contrast, when the recombinant enzyme treatment was applied, the enzyme enabled expression of eGFP by recognizing two loxP sites in the vector and removing the DsRed sequence.

TAT-Cre mediated recombination activity in 293FT cells and cytotoxicity to TAT-Cre were evaluated, and evaluation was performed at various doses and durations for TAT-Cre treatment (see FIGS. 4C and 5 ). After 24 hours incubation, significant cytotoxicity to TAT-Cre was observed at 4 or 6 μM (FIG. 4C). When cells transfected with TAT-Cre were treated, a green fluorescent signal due to eGFP expression (a result of TAT-Cre activity) appeared 12 hours after TAT-Cre was added, whereas a red fluorescent signal from DsRed expression was transfected. It lasted for 12 hours immediately afterwards. Green fluorescence increased when TAT-Cre was incubated for 48 hours, and the signal was proportional to the concentration for TAT-Cre during treatment. On the other hand, after 48 hours treatment, red fluorescence decreased, and the fluorescent signal was inversely proportional to the concentration of TAT-Cre during treatment, and the in vivo recombination was successful and was dependent on time and dose (see FIG. 5). These results suggest that the addition of a polyglutamate tag to TAT-Cre does not inhibit the activity of TAT and NLS signals and Cre recombination for expression and purification in E. coli.

(4) Discussion

It is considered a safer and more useful method of delivery because the delivery of proteins is less toxic and more efficient for transduction than delivery of genetic material. CPP enables high-efficiency protein delivery, and CPP fusion proteins are used for therapeutic purposes or for protein delivery and have been commercialized in some cases. Among CPP, TAT peptides have been used for protein delivery because they can pass through the plasma membrane of all types of mammalian cells except Caco-2 and MDCK cells. Together with NLS, the cargo protein can be transported to the nucleus through the nuclear pore. Significant amounts of TAT fusion proteins expressed in E. coli or other bacterial hosts often form inclusion bodies. Researchers have tried to reduce the amount of recombinant protein accumulated in the inclusion bodies in a variety of ways, including optimization of the culture temperature and induction conditions.

In the present invention, in order to overcome the accumulation of the recombinant TAT-Cre protein in the inclusion body, a negatively charged peptide tag was inserted near the positively charged TAT sequence to improve the solubility of the cargo protein in the E. coli expression system. It was confirmed that neutralization of positively charged TAT and highly cationic NLS by polar glutamate (E12) reduced the accumulation of recombinant proteins and increased solubility in the inclusion bodies.

It was found that TAT-Cre expression increases the amount of total and soluble protein and decreases the amount of insoluble protein (see Table 1). The reason why the amount of total protein is higher may be due to the increased biomass of E12-TAT-Cre-expressed E. coli (see FIG. 3A). This phenomenon may be due to a reduction in toxicity which may be associated with high cationicity for the TAT and NLS amino acid sequences. In contrast to other reports suggesting that expression of the general TAT-Cre protein increases the accumulation of Cre in the inclusion bodies, the present inventors have confirmed the substantial expression of soluble recombinant TAT-Cre in E. coli. Insertion of the E12 domain further improved the solubility and yield of the recombinant TAT-Cre. Whether this strategy is applicable to other proteins can be further confirmed.

Although TAT peptide is less toxic than amphiphilic CPP, TAT-Cre exhibited cytotoxicity at high concentration (see FIG. 4C). However, this problem is due to the toxicity of Cre, not TAT. Prolonged expression of Cre has cardiotoxicity, and if the expression of Cre is high enough to affect cell physiology, Cre alone can exhibit cytotoxicity. Since inserting TAT peptides into proteins of interest can be efficiently delivered into cells, this strategy has been applied to alleviate neurotoxicity, promote differentiation in pancreatic beta cells, and immunotherapy in Parkinson's disease mouse models. Thus, arginine-rich CPPs, including TAT, have a wide range of applications for research and therapeutic purposes.

Once the E12 sequence along with the His tag is removed by TEV protease during the purification process, the resulting TAT sequence is fully exposed and functional as demonstrated by cell-based assays. It was found that the TAT-Cre recombinase activity did not perform complete recombination in a cell-free system (see Fig. 4B). This phenomenon is reported in another report [S.K. Lyu, et al., BMC biotechnology 15 (2015) 7 and P. Oberdoerffer, et al., Nucleic Acids Research 31 (2003) e140].

In contrast, when evaluating activity in mammalian cells with phloxed alleles, the recombination was carried out enzymatically in a dose and time dependent manner (see Fig. 5). When mortality is caused by tissue-specific gene knockout after Cre-loxP recombination in genetically engineered phloxed mice, TAT-Cre can be used for subsequent studies of the mechanism. To prevent the above lethality, a tamoxifen-induced Cre system was devised. Another objective of the inducible system is to prevent chronic effects of gene deletion in the embryonic period. Nevertheless, administration of tamoxifen causes side effects as well as induction of Cre. The application of TAT-Cre can prevent such toxicity, and the in-depth mechanism can be explained in depth through manipulation of primary cells isolated from phloxed mice.

(5) Conclusion

In the present invention, it is reported that the solubility and yield of the recombinant protein obtained by adding a polyglutamate domain to TAT-Cre and expressing it in E. coli can be improved. In addition, it was found that the growth rate of E. coli expressing E12-TAT-Cre is increased by neutralizing the positive charge of the TAT and NLS sequences. As a result, it was confirmed that TAT-Cre is functional in cell and cell-free systems. These results suggest that the addition of the polyglutamate domain is a new method to improve the expression and purification of the target TAT fusion protein.

(6) Green

Cre recombinase is widely used to manipulate DNA sequences for in vitro and in vivo studies. By inserting a trans-activator of transcription (TAT) sequence into Cre, TAT-Cre can pass through the cell membrane, and NLS (nuclear localization signal) is added to allow the enzyme to translocate to the nucleus. It helps. Since the yield of recombinant TAT-Cre is limited by the formation of inclusion bodies, it was hypothesized that the positively charged arginine-rich TAT sequence forms inclusion bodies, whereas neutralization by the addition of a negatively charged sequence improves the solubility of the protein. . To demonstrate this, the positively charged TAT sequence was neutralized by proximal insertion of a negatively charged polyglutamate (E12) sequence.

When the E12 tag was expressed in E. coli, it was confirmed that the solubility and yield of E12-TAT-NLS-Cre (E12-TAT-Cre) were improved compared to TAT-NLS-Cre (TAT-Cre). In addition, the growth of cells expressing E12-TAT-Cre was increased compared to that of cells expressing TAT-Cre. The efficacy of purified TAT-Cre was confirmed by recombination tests on plotted plasmids in cell-free systems and 293FT cells.

Taken together, it was found that inserting the E12 sequence into TAT-Cre improves the solubility of TAT-Cre during expression in E. coli (possibly neutralizing the ion-charge effect of the TAT sequence), and consequently increases the yield. These methods can be applied to the production of convertible proteins for research and therapeutic purposes.

<110> Gachon University of Industry-Academic cooperation Foundation <120> Method for preparing polyglutamate-TAT-Cre fusion protein <130> P190202 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> polyglutamate <400> 1 Glu Glu Glu Glu Glu Gly Ser Glu Glu Glu Glu Glu Glu Glu 1 5 10

Claims (18)

(a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene;
(b) expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein; And
(c) purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b)
A method for producing a polyglutamate-TAT-Cre fusion protein comprising a. The method of claim 1, wherein the polyglutamate-TAT-Cre gene of step (a) further comprises any one of a His tag and a GST tag. The method of claim 1, wherein the polyglutamate-TAT-Cre gene of step (a) further comprises a TEV protease recognition site. The method of claim 1, wherein the polyglutamate-TAT-Cre gene of step (a) is at the N-terminus of the TAT peptide in the group consisting of glycine (G), methionine (M), and alanine (A). A method for producing a polyglutamate-TAT-Cre fusion protein, characterized in that it further comprises at least one selected amino acid residue. The method of claim 1, wherein the expression is induced to 0.05 to 5 mM IPTG in step (b). The method for producing a polyglutamate-TAT-Cre fusion protein according to claim 5, wherein the IPTG is induced in the host cell for 1 to 5 hours. The method of claim 1, wherein the expression in step (b) is performed using E. coli as a host cell. (a) inserting a polyglutamate sequence consisting of the amino acid sequence of SEQ ID NO: 1 into a gene encoding TAT-Cre to obtain a polyglutamate-TAT-Cre gene;
(b) expressing the polyglutamate-TAT-Cre gene obtained in step (a) to obtain a polyglutamate-TAT-Cre recombinant protein;
(c) purifying the polyglutamate-TAT-Cre recombinant protein obtained in step (b);
(d) reacting the polyglutamate-TAT-Cre recombinant protein obtained in step (c) with TEV protease to obtain a product; And
(e) purifying the product obtained in step (d) to obtain TAT-Cre protein
Method for producing a TAT-Cre protein comprising a. The method of claim 8, wherein the reaction in step (d) is carried out at 25 to 35°C. The method of claim 8, wherein the reaction in step (d) is performed at a pH of 7.0 to 9.0. The polyglutamate-TAT-Cre fusion protein prepared according to any one of claims 1 to 7. The polyglutamate-TAT-Cre fusion protein according to claim 11, wherein the protein is 46 kDa. The TAT-Cre protein produced by any one of claims 8 to 10. 14. The TAT-Cre protein according to claim 13, wherein the protein is 42 kDa. The polyglutamate-TAT-Cre fusion protein of claim 11; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is 1 selected from the group consisting of proteins, nucleic acids, peptides, lipids, glycolipids, minerals, sugars, contrast substances, drugs and chemicals. More than a species, cargo delivery system. A pharmaceutical composition for cell penetration comprising the polyglutamate-TAT-Cre fusion protein of claim 11. The TAT-Cre protein of claim 13; And a cargo fused to the N-terminus or C-terminus of the fusion protein, wherein the cargo is one selected from the group consisting of proteins, nucleic acids, peptide peptides, glycolipids, minerals, sugars, contrast substances, drugs, and chemical substances. What is more, the cargo delivery system. A pharmaceutical composition for cell penetration comprising the TAT-Cre protein of claim 13.

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