WO2019181235A1

WO2019181235A1 - Method for producing lipidated protein, and lipidated protein

Info

Publication number: WO2019181235A1
Application number: PCT/JP2019/003695
Authority: WO
Inventors: 典穂神谷; 里衣若林; 孝介南畑; 茉莉 ▲高▼原
Original assignee: 国立大学法人九州大学
Priority date: 2018-03-19
Filing date: 2019-02-01
Publication date: 2019-09-26
Also published as: JPWO2019181235A1

Abstract

The present disclosure provides a method for producing a lipidated protein, said method comprising a step for reacting a fused protein with a compound having a primary amino group in the presence of a transglutaminase derived from a microorganism to thereby give a lipidated protein in which the fused protein is crosslinked to the compound via an isopeptide bond, wherein the fused protein comprises a first peptide having a glutamine residue and a protein while the compound has a lipid moiety or a fat-soluble vitamin moiety.

Description

Method for producing lipidated protein and lipidated protein

The present invention relates to a method for producing a lipidated protein and a lipidated protein.

Lipidated proteins play an important role in the process of cell function expression and function control. Analyzing the function of lipidated proteins on cell membranes is a useful tool for analyzing various diseases. Therefore, analysis of the function of lipidated proteins on the cell membrane has attracted attention. Lipidated proteins also have properties such as being able to be anchored to a drug delivery carrier. Therefore, application of lipidated proteins as pharmaceuticals is also expected.

Conventionally, protein lipidation methods have problems such as low reaction efficiency and site specificity, and lower functions of the resulting lipidated protein compared to functions expected of the starting protein. As a site-specific and efficient lipid modification means, a method for producing a lipidated protein using a microorganism-derived transglutaminase has been studied. For example, Non-Patent Document 1 discloses a fusion protein in which a peptide containing a lysine residue is bound to the C-terminus of a green fluorescent protein in the presence of a microorganism-derived transglutaminase, and a fatty acid at the N-terminus of a peptide having a glutamine residue. A method of obtaining a lipidated protein by reacting with a lipid peptide to which is bound is described.

Depending on the combination of the fusion protein and lipid peptide used to obtain the lipidated protein, the transglutaminase reaction may not proceed easily, and the substrate for the transglutaminase reaction may be limited. Any method for producing a lipidated protein capable of easily carrying out a transglutaminase reaction regardless of the type of protein and lipid is useful.

Therefore, the present invention can introduce various lipids or fat-soluble vitamins corresponding to uses required for lipidated proteins and the like, and a lipidated protein production method and lipidation capable of easily producing lipidated proteins The object is to provide a protein.

In one aspect of the present invention, a lipidated protein in which a fusion protein and a compound having a primary amino group are reacted in the presence of a microorganism-derived transglutaminase, and the fusion protein and the compound are cross-linked by an isopeptide bond. Wherein the fusion protein has a first peptide and protein containing a glutamine residue, and the compound has a lipid part or a fat-soluble vitamin part. .

In conventional methods, there are cases where the lipid peptide obtained by the reaction of a peptide having a glutamine residue with a fatty acid has high hydrophobicity and the reactivity in the transglutaminase reaction is lowered. In such a case, a method for improving the water solubility of the lipid peptide by introducing a hydrophilic amino acid residue into the peptide having a glutamine residue is considered. However, when a hydrophilic amino acid residue is introduced into a peptide containing a glutamine residue, it tends to be difficult to be recognized as a substrate by a microorganism-derived transglutaminase.

If the lipid peptide is difficult to dissolve in water, it can be considered that the lipid peptide is dissolved by adding a surfactant, an organic solvent or the like to the reaction system. However, since it is possible to induce a decrease or disappearance of the enzyme activity of the microorganism-derived transglutaminase, there is a concern that it may be difficult to obtain a lipidated protein. In view of application to pharmaceuticals and the like, it is preferable to refrain from using organic solvents as much as possible in the production of lipidated proteins.

In the method for producing lipidated protein, contrary to the conventional method, the fusion protein containing the protein to be lipidated has a glutamine residue, and the compound containing lipid has a primary amino group. . Microorganism-derived transglutaminase can recognize a fusion protein and a compound having a primary amino group as a substrate. In addition, by introducing a primary amino group as a functional group capable of imparting hydrophilicity to the lipid side, the transglutaminase reaction proceeds between the fusion protein and the compound having the primary amino group, and the lipid can be easily Protein can be produced.

The compound may include a second peptide containing a lysine residue and a lipid peptide having a lipid part or a fat-soluble vitamin part, and the primary amino group may be derived from the lysine residue. . The second peptide containing a lysine residue and the lipid peptide having a lipid part or a fat-soluble vitamin part are easily recognized as a substrate by the microorganism-derived transglutaminase. When the above compound contains such a lipid peptide, a lipidated protein can be produced more easily. Moreover, since the said lipid peptide has a peptide chain (2nd peptide), the kind and number of amino acid residues of a 2nd peptide part can be adjusted. Thus, for example, the type and number of amino acid residues can be adjusted according to the lipid to be introduced into the protein. When the compound contains a lipid peptide, options for lipids or fat-soluble vitamins that can be post-modified to proteins by the above production method can be expanded.

The second peptide may have a higher ratio of hydrophilic amino acid residues than the first peptide. By making the ratio of the hydrophilic amino acid residue of the second peptide higher than that of the first peptide, it is possible to weaken the degree of hydrophobicity derived from the lipid portion of the lipid peptide and improve water solubility. The transglutaminase reaction can be made easier to proceed.

The second peptide may further contain at least one amino acid residue selected from the group consisting of a histidine residue, a proline residue, a tryptophan residue, and an arginine residue. When the second peptide contains the amino acid residue, the lipid peptide is more easily recognized by the microorganism-derived transglutaminase, and the transglutaminase reaction can be further promoted.

The lipid part may contain an aliphatic hydrocarbon group having 12 to 18 carbon atoms. When the lipid part has the above-described configuration, the obtained lipidated protein is easily anchored by the cell membrane.

The first peptide is at least one hydrophobic group selected from the group consisting of glycine residues, alanine residues, valine residues, leucine residues, isoleucine residues, tyrosine residues, tryptophan residues, and phenylalanine residues. An amino acid residue may be further included. When the first peptide contains the hydrophobic amino acid residue, the first peptide in the fusion protein can be more easily recognized by the microorganism-derived transglutaminase, and the transglutaminase reaction can proceed more easily.

In another aspect, the present invention includes a fusion protein part having a first peptide and protein containing a glutamine residue, and a lipid part or a fat-soluble vitamin part, wherein the lipid part or the fat-soluble vitamin part is the first part. Provided is a lipidated protein which is bound to the fusion protein part by an isopeptide bond with a glutamine residue of one peptide.

The above lipidated protein is easily synthesized because the fusion protein part has a glutamine residue, and the lipid part or fat-soluble vitamin part has a structure in which the fusion protein part is bound to the glutamine residue by an isopeptide bond. can do.

The lipid part or the fat-soluble vitamin part includes a lipid peptide part having a second peptide containing a lysine residue, and the isopeptide bond is a bond between the lysine residue and the glutamine residue. Also good.

The second peptide may have a higher ratio of hydrophilic amino acid residues than the first peptide.

The second peptide may further contain at least one amino acid residue selected from the group consisting of a histidine residue, a proline residue, a tryptophan residue, and an arginine residue.

The lipid part may have an aliphatic hydrocarbon group having 12 to 18 carbon atoms. When the lipid has the above-described configuration, the lipidated protein is more easily anchored to the cell membrane.

The first peptide further contains at least one hydrophobic amino acid residue selected from the group consisting of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue and a phenylalanine residue. Also good.

According to the present invention, it is possible to introduce various lipids or fat-soluble vitamins corresponding to required uses and the like, and to provide a lipidated protein production method and a lipidated protein capable of easily producing a lipidated protein. be able to.

FIG. 1 is a graph showing the results of a cytotoxicity test of a synthesized lipid peptide. FIG. 2 is a graph showing the results of measuring the fluorescence intensity ratio (I ₃₈₅ / I ₃₇₃ ) of the fluorescent substance (pyrene) encapsulated in the synthesized lipid peptide. FIG. 3 is a graph showing the results of circular dichroism spectrum measurement of the synthesized lipid peptide. FIG. 4 is a graph showing the results of circular dichroism spectrum measurement of the synthesized lipid peptide. FIG. 5 is a liquid chromatograph showing the reaction results of the fusion protein and lipid peptide in Example 1. FIG. 6 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Examples 1 and 2. FIG. 7 is a graph showing the relationship between the reaction rate of the transglutaminase reaction and the reaction time in Examples 1 and 2. FIG. 8 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Examples 3 and 4. FIG. 9 is a graph showing the relationship between the transglutaminase reaction rate and reaction time in Examples 3 and 4. FIG. 10 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Examples 5 and 6. FIG. 11 is a graph showing the relationship between the reaction rate of the transglutaminase reaction and the reaction time in Example 6. FIG. 12 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Example 3, Example 4, and Examples 7 to 10. FIG. 13 is a graph showing the relationship between the reaction rate of the transglutaminase reaction and the reaction time in Example 3, Example 4, and Examples 7 to 10. FIG. 14 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Example 3, Example 4, and Examples 11-16. FIG. 15 is a graph showing the relationship between the reaction rate of the transglutaminase reaction and the reaction time in Example 3, Example 4, and Examples 11 to 16. FIG. 16 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 3, Example 4, and Examples 8 to 10 were applied to floating cells. is there. FIG. 17 is a graph showing cytotoxicity evaluation results after applying the lipidated protein and fusion protein obtained in Example 3, Example 4, and Examples 8 to 10 to floating cells. FIG. 18 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 4 and Examples 11 to 16 were applied to floating cells. FIG. 19 is a graph showing cytotoxicity evaluation results after the lipidated protein and fusion protein obtained in Example 3, Example 4, and Examples 11 to 16 were applied to floating cells. FIG. 20 is a graph showing the relationship between the transglutaminase reaction rate and the K / Q ratio in Examples 25-28. FIG. 21 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Examples 21 to 22 and 25 to 28 were applied to floating cells. FIG. 22 is a fluorescence micrograph showing a state where the lipidated protein and fusion protein obtained in Examples 25 to 28 are applied to floating cells. FIG. 23 is a fluorescence micrograph showing a state in which the lipidated protein and fusion protein obtained in Example 28 were applied to floating cells. FIG. 24 is a graph showing the cytotoxicity evaluation results after the lipidated protein and fusion protein obtained in Examples 21 and 22 were applied to floating cells. FIG. 25 is a graph showing cytotoxicity evaluation results after the lipidated protein and fusion protein obtained in Examples 26 to 28 were applied to floating cells. FIG. 26 is a graph showing the results of a cytotoxicity test of the synthesized lipid peptide. FIG. 27 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 9 and Examples 29 to 34 were applied to floating cells. FIG. 28 is a graph showing the cytotoxicity evaluation results after applying the lipidated protein and fusion protein obtained in Example 4 and Examples 29 to 34 to floating cells. FIG. 29 is a graph showing the results of a cytotoxicity test of the synthesized lipid peptide. FIG. 30 is a graph showing the results of cytotoxicity tests of synthesized lipid peptides. FIG. 31 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 11 and Examples 42 to 47 were applied to floating cells. FIG. 32 is a graph showing the cytotoxicity evaluation results after the lipidated protein and fusion protein obtained in Example 11 and Examples 42 to 47 were applied to floating cells. FIG. 33 is a graph showing the results of cytotoxicity tests of synthesized lipid peptides. FIG. 34 is a graph showing the results of a cytotoxicity test of the synthesized lipid peptide. FIG. 35 is a graph showing the results of a cytotoxicity test of the synthesized lipid peptide. FIG. 36 is a graph showing the relationship between the reaction rate of the synthesized lipid peptide transglutaminase reaction and the K / Q ratio. FIG. 37 is a graph showing the relationship between the reaction rate of the synthesized lipid peptide transglutaminase reaction and the K / Q ratio. FIG. 38 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 16 and Examples 55 to 64 were applied to floating cells. FIG. 39 is a graph showing the evaluation results of cytotoxicity after applying the lipidated protein and fusion protein obtained in Examples 55 to 57, Examples 59 to 63, and Example 66 to floating cells. FIG. 40 is a graph showing the amount of fluorescence generated per cell after the lipidated protein and fusion protein obtained in Example 67 were applied to floating cells. FIG. 41 is a graph showing the evaluation results of cytotoxicity after the lipidated protein and fusion protein obtained in Example 68 were applied to floating cells.

Hereinafter, embodiments of the present invention will be described with reference to the drawings as the case may be. However, the following embodiments are examples for explaining the present invention, and are not intended to limit the present invention to the following contents.

[Method for producing lipidated protein]
In one embodiment of a method for producing a lipidated protein, a fusion protein and a compound having a primary amino group are reacted in the presence of a microorganism-derived transglutaminase, and the fusion protein and the compound are cross-linked by an isopeptide bond. Obtaining a lipidated protein obtained. The fusion protein has a first peptide and protein containing a glutamine residue. The compound has a lipid part or a fat-soluble vitamin part.

In the method for producing a lipidated protein according to this embodiment, a fusion protein having a first peptide containing a glutamine (Q) residue and a compound having a primary amino group are both recognized as substrates by a microorganism-derived transglutaminase. Therefore, an isopeptide bond is formed by a transglutaminase reaction, and a lipidated protein into which a lipid or a fat-soluble vitamin is easily introduced can be obtained.

The number of amino acid residues constituting the first peptide of the fusion protein may be 1 to 27, 2 to 27, 2 to 10, or 2 to 8. Or 2-7. The first peptide may be, for example, a peptide including an amino acid sequence represented by the X ¹ _n QX ² _m motif. Here, n may be an integer of 0 to 13, an integer of 1 to 13, an integer of 1 to 9, or an integer of 1 to 4. . m may be an integer of 0 to 13, an integer of 1 to 13, an integer of 1 to 9, or an integer of 1 to 4. In the X ¹ _n QX ² _m motif, a plurality of X ¹ and a plurality of X ² may be the same as or different from each other.

In the X ¹ _n QX ² _m motif, X ¹ represents an amino acid residue other than a glutamine residue and a lysine (K) residue. ^{X 1} in X ¹ _n QX ² _m motif, glycine (G), alanine (A) residue, valine (V), leucine (L) residues, isoleucine (I) residue, a tyrosine (Y ) Residue, proline (P) residue, tryptophan (W) residue, phenylalanine (F) residue, histidine (H) residue, and at least one amino acid residue selected from the group consisting of arginine (R) residues Or at least one hydrophobic group selected from the group consisting of glycine residue, alanine residue, valine residue, leucine residue, isoleucine residue, tyrosine residue, tryptophan residue, and phenylalanine residue Sex amino acid residues may be included.

^{X 2} in X ¹ _n QX ² _m motif shows the amino acid residue other than glutamine and lysine residues. ^{X 2} in X ¹ _n QX ² _m motif, for example, methionine (M) residues, arginine (R) residue, at least one amino acid residue selected from the group consisting of glycine and serine (S) residues Groups may be included.

The amino acid sequence represented by the X ¹ _n QX ² _m motif may be, for example, an amino acid sequence represented by FYPLQMRG, LLQG, AWHRPQFGG, or the like.

The protein constituting the fusion protein is not particularly limited, and examples thereof include green fluorescent protein, antibody, enzyme, antigen protein, growth factor, and hormones (for example, insulin and growth hormone) that act on physiological activity. Can be mentioned.

The first peptide may be directly bonded to the C-terminal or N-terminal of the protein, or may be bonded to the first linker by binding a first linker to the C-terminal or N-terminal of the protein. That is, the fusion protein may have a protein, a first linker, and a first peptide. When the fusion protein has a first linker, the fusion protein may include, for example, a fusion protein represented by [protein]-[first linker]-[first peptide] A fusion protein represented by [peptide]-[first linker]-[protein] may be included.

The first linker contains 1 to 20 amino acid residues. The amino acid residue constituting the first linker may include at least one amino acid residue selected from the group consisting of a glycine residue, a serine residue, and a proline residue. It may contain at least one amino acid residue selected from the group consisting of Here, the number of amino acid residues constituting the first linker is determined as follows. First, the amino acid residues of the fusion protein are analyzed, and the number of amino acid residues is counted from the amino acid residue directly connected to the C-terminal or N-terminal of the protein constituting the fusion protein toward the first peptide. When amino acid residues other than glycine residue, serine residue and proline residue are detected before the number of amino acid residues reaches 20, the number of amino acid residues up to the detected amino acid residue The number obtained by subtracting one from is used as the number of amino acid residues constituting the first linker.

The first linker is, for example, a peptide represented by a G _r motif, a (G _r S) _s motif, a (PG _r ) _s motif, a (PG _r S) _s motif, and a (SG _r S) _s motif. It may be. Here, r is an integer of 1-6. Here, s is an integer of 1 to 5. More specifically, examples of the first linker include GGGS, PGGG, SGGGS, and PGGGS.

The fusion protein may be obtained by chemical synthesis, or may be obtained by expressing the protein using a transformed host. As the fusion protein obtained by chemical synthesis, for example, a protein obtained by introducing the first peptide or the first peptide and the first linker by post-expression modification of the protein may be used. As a fusion protein obtained using a transformed host, for example, a protein in which the first peptide or the amino acid sequence of the first peptide and the first linker is introduced into the amino acid sequence of the protein (modified protein) Designing and expressing the modified protein by a host transformed with an expression vector having a nucleic acid sequence encoding the modified protein and one or more regulatory sequences operably linked to the nucleic acid sequence. You may use what is obtained. Here, the regulatory sequence, expression vector, host and the like are not particularly limited.

The method for producing the nucleic acid encoding the modified protein is not particularly limited, and may be a method of cloning by amplification using polymerase chain reaction (PCR) using the designed gene, or a method of producing by chemical synthesis. May be. In order to facilitate purification of the modified protein, etc., a nucleic acid encoding a modified protein comprising an amino acid sequence in which an amino acid sequence comprising a start codon and a His tag is added to the N-terminus of the amino acid sequence of the modified protein may be synthesized. .

The compound having a primary amino group has a lipid part or a fat-soluble vitamin part. Examples of the compound having a primary amino group and a lipid part include aliphatic amines such as tetradecylamine and hexadecylamine. The compound having a primary amino group may be, for example, a second peptide containing a lysine residue and a lipid peptide having a lipid part or a fat-soluble vitamin part. In this case, the primary amino group may be an amino group derived from a lysine residue. The compound is, for example, a lipid peptide having a lysine residue and a lipid part or a fat-soluble vitamin part. When the above compound has a lysine residue, it is more easily recognized as a substrate by the microorganism-derived transglutaminase.

The second peptide of the compound may be composed of only lysine residues or may be composed of a plurality of amino acid residues. The number of amino acid residues constituting the second peptide may be 1 to 27, 2 to 27, 2 to 10, 2 to 8, or It may be 2-7. The second peptide may be, for example, a peptide including an amino acid sequence represented by the X ³ _p KX ⁴ _q motif. Here, p may be an integer of 0 to 13, an integer of 1 to 13, an integer of 1 to 9, or an integer of 1 to 4. . q may be an integer of 0 to 13, an integer of 1 to 13, an integer of 1 to 9, or an integer of 1 to 4. In the X ³ _p KX ⁴ _q motif, a plurality of X ³ and a plurality of X ⁴ may be the same as or different from each other.

In the X ³ _p KX ⁴ _q motif, X ³ represents an amino acid residue other than a glutamine residue and a lysine residue. X ³ in X ³ _p KX ⁴ _q motif preferably comprises a histidine residue, a proline residue, at least one amino acid residue selected from the group consisting of tryptophan and arginine residues, more preferably, It contains at least one basic amino acid residue selected from the group consisting of histidine residues and arginine residues. By X ³ _p KX ⁴ _q X in motif ³ includes the amino acid residue, the reaction solution of the lipid peptide (e.g., aqueous solution) it is possible to improve the solubility in the available lipid or While expanding the choice of fat-soluble vitamins, it can further promote the progress of the transglutaminase reaction.

^{X 4} in the X ³ _p KX ⁴ _q motif shows the amino acid residue other than glutamine and lysine residues. X ³ _p X ⁴ in KX ⁴ _q motif, for example, may include at least one amino acid residue selected from the group consisting of glycine and serine residues.

The amino acid sequence represented by the X ³ _p KX ⁴ _q motif may be, for example, an amino acid sequence represented by MRHKGS, KGS, RKGS, HKGS, or the like. The amino acid sequence represented by the X ³ _p KX ⁴ _q motif may also be, for example, K, RK, HK and RHK.

The lipid part is a part having a structure derived from lipid. The fat-soluble vitamin part is a part having a structure derived from a fat-soluble vitamin. The lipid includes a fatty acid, a steroid and the like. Examples of fatty acids include saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. Examples of steroids include cholesterol. Examples of cholesterol include lithocholic acid and the like. The lipid may be, for example, a lipid having at least one hydrocarbon group selected from the group consisting of aliphatic hydrocarbon groups, and the aliphatic hydrocarbon group may be linear, branched or alicyclic You may have. The aliphatic hydrocarbon group may be, for example, a lipid having an aliphatic hydrocarbon group having 6 or more carbon atoms, a lipid having an aliphatic hydrocarbon group having 12 to 29 carbon atoms, and 12 to 12 carbon atoms. It may be a lipid having 18 aliphatic hydrocarbon groups. When the lipid has a hydrocarbon group as described above, the tethered lipidated protein to the cell membrane can be made stronger, and the toxicity to the cell can be made sufficiently low. Examples of the lipid having an aliphatic hydrocarbon group having 12 to 18 carbon atoms include saturated fatty acids such as lauric acid, myristic acid, palmitic acid, and stearic acid. Examples of the lipid having an aliphatic hydrocarbon group having 12 to 18 carbon atoms include unsaturated fatty acids such as oleic acid and linoleic acid in addition to the saturated fatty acid. Examples of the lipid having an aliphatic hydrocarbon group having 12 to 24 carbon atoms include lithocholic acid and the like in addition to the saturated fatty acid. Examples of the fat-soluble vitamin include vitamin A (such as retinal), vitamin D (such as ergocalciferol and cholecalciferol), vitamin E (such as α-tocopherol), and vitamin K (phylloquinone, menaquinone, menadione, and Menadiol, etc.).

The second peptide may be directly bonded to the carboxyl group or amino group at the end of the lipid part or fat-soluble vitamin part, and the second peptide may be bonded to the carboxyl group or amino group at the end of the lipid part or fat-soluble vitamin part. A linker may be bound to the second linker. That is, the lipid peptide may have a lipid part or a fat-soluble vitamin part, a second linker, and a second peptide. When the lipid peptide has a second linker, the lipid peptide may include, for example, a lipid peptide represented by [lipid part]-[second linker]-[second peptide] or [lipid A lipid peptide represented by [soluble vitamin part]-[second linker]-[second peptide] may be included.

The second linker may be a linker peptide containing 1 to 10 amino acid residues, or may be a polyalkylene glycol chain. The amino acid residue constituting the linker peptide may include at least one amino acid residue selected from the group consisting of a glycine residue, a serine residue and a proline residue. Here, the number of amino acid residues constituting the linker peptide is determined as follows. First, the amino acid residue of the peptide part which comprises a lipid peptide is analyzed, and the number of amino acid residues is counted toward the 2nd peptide from the amino acid residue directly connected to a lipid part or a fat-soluble vitamin part. When amino acid residues other than glycine residue, serine residue and proline residue are detected before the number of amino acid residues reaches 10, the number of amino acid residues up to the detected amino acid residue The number obtained by subtracting one from is used as the number of amino acid residues constituting the linker peptide.

The amino acid residue constituting the linker peptide may also contain at least one amino acid residue selected from the group consisting of lysine residue, glycine residue, serine residue and proline residue. In this case, the number of amino acid residues constituting the linker peptide is determined as follows. First, the amino acid residue of the peptide part which comprises a lipid peptide is analyzed, and the number of amino acid residues is counted toward the 2nd peptide from the amino acid residue directly connected to a lipid part or a fat-soluble vitamin part. When amino acid residues other than lysine residues, glycine residues, serine residues and proline residues are detected before the number of amino acid residues reaches 10, from the number of detected amino acid residues The number reduced by one is taken as the number of amino acid residues constituting the linker peptide.

The second linker is, for example, a peptide represented by a G _r motif, a (G _r S) _s motif, a (PG _r ) _s motif, a (PG _r S) _s motif, and a (SG _r S) _s motif. It may be. Here, r represents an integer of 1 to 6. Here, s represents an integer of 1 to 5. More specifically, examples of the second linker include those having an amino acid sequence such as GG, GGGGS, SGGGS, PGGG, and PGGGS. The polyalkylene glycol chain is represented by (RO) _t . Here, t represents an integer of 1 to 3, and R represents an alkyl group. Examples of the polyalkylene glycol chain include polyethylene glycol and polypropylene glycol.

As the above compound (for example, the above lipid peptide), for example, a compound obtained by amination of a lipid terminal or the like may be used, or a compound obtained by peptide synthesis may be used. For peptide synthesis, for example, the Fmoc solid phase synthesis method can be used. The resin used for the Fmoc solid phase synthesis method can be selected according to the characteristics required for the lipid peptide, the characteristics of the fusion protein to be reacted, and the like.

The first peptide may have a higher proportion of hydrophobic amino acid residues than the second peptide. By setting the first peptide and the second peptide as described above, the fusion protein and the lipid peptide can be easily recognized by the microorganism-derived transglutaminase.

The second peptide may have a higher proportion of hydrophilic amino acid residues than the first peptide. By setting the first peptide and the second peptide as described above, the degree of hydrophobicity derived from the lipid part or the fat-soluble vitamin part of the lipid peptide can be weakened and the transglutaminase reaction can be promoted.

The critical micelle concentration of the compound (eg, the lipid peptide) may be, for example, 1 μM or more, 5 μM or more, 10 μM or more, or 20 μM or more. The critical micelle concentration of the compound (eg, the lipid peptide) may be, for example, 350 μM or less, 300 μM or less, 250 μM or less, or 200 μM or less. The critical micelle concentration of the compound (eg, the lipid peptide) may also be, for example, 150 μM or less, or 140 μM or less. When the critical micelle concentration of the compound is within the above range, the compound is prevented from forming micelles in the system during the transglutaminase reaction, and the compound enters the reaction site of the microorganism-derived transglutaminase. And the transglutaminase reaction can be promoted. The critical micelle concentration can be adjusted, for example, by selecting the type of lipid part or fat-soluble vitamin part, the length of the second linker and second peptide, the type of amino acid residue constituting the part, and the like. The critical micelle concentration was determined by measuring the ratio (I ₃₈₅ / I ₃₇₃ ) of the fluorescence intensity (I ₃₈₅ ) at ₃₈₅ nm and the fluorescence intensity (I ₃₇₃ ) at ₃₇₃ nm using a solution containing pyrene in the above compound. It can be determined from the change in the ₃₈₅ / I ₃₇₃ ratio. Specifically, it can be determined based on the method described in Examples described later.

The compound (for example, the lipid peptide) can have various secondary structures depending on the type of lipid part or fat-soluble vitamin part. Examples of the secondary structure of the compound include a β sheet structure and a random coil structure. For example, when the secondary structure of the compound is a β sheet structure, the ability to anchor to the cell membrane can be improved.

The microorganism-derived transglutaminase may be a wild-type microorganism-derived transglutaminase or a mutant of a wild-type microorganism-derived transglutaminase. In the present specification, “a mutant of a wild-type microorganism-derived transglutaminase” refers to an amino acid sequence modified from a wild-type microorganism-derived transglutaminase or a wild-type microorganism-derived transglutaminase. It is a protein that is artificially designed and synthesized regardless of the amino acid sequence and has transglutaminase activity.

The mutant of the wild-type microorganism-derived transglutaminase is, for example, a substitution, deletion, insertion and / or substitution of one or more amino acid residues in the amino acid sequence of the wild-type microorganism-derived transglutaminase (excluding the signal peptide portion). It has an amino acid sequence corresponding to the addition. Substitution, deletion, insertion and / or addition of amino acid residues can be performed by methods well known to those skilled in the art such as partial-directed mutagenesis. Substitution, deletion, insertion and / or addition of amino acid residues is, for example, 1 to 10 amino acid residues, 1 to 5 amino acid residues, 1 to 3 amino acid residues, 1 amino acid residue It may be performed on a group. Specifically, a mutant of a microorganism-derived transglutaminase disclosed in International Publication No. 2018/004014 can be used.

In the method for producing lipidated protein, a reaction is performed in a reaction solution in which a microorganism-derived transglutaminase, a fusion protein, and a compound having a primary amino group (for example, a compound containing a lipid peptide) are dissolved in a solvent. The solvent includes water, and for example, phosphate buffered saline can be used. The reaction temperature can be adjusted according to the activity of the microorganism-derived transglutaminase. For example, the reaction temperature may be 50 ° C. or higher, 60 ° C. or higher, and 70 ° C. or lower.

The concentration of the fusion protein and the compound containing the lipid peptide in the reaction solution can be adjusted by the type and combination of the compound containing the fusion protein and the lipid peptide. For example, the molar ratio of the primary amino group (here lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein ( The (K / Q ratio) may be, for example, 3 or more, 4 or more, 5 or more, 10 or more, or 15 or more. The K / Q ratio may be, for example, 25 or less or 20 or less. For example, lipids can be more efficiently introduced into proteins by setting the K / Q ratio within the above numerical range.

The method for producing a lipidated protein can also be performed in the presence of a target cell on which the lipidated protein acts. That is, another embodiment of the method for producing a lipidated protein includes a compound having a primary amino group and a fusion protein in the presence of a cell (for example, a cell to be treated with a lipidated protein) and a microorganism-derived transglutaminase. To obtain a lipidated protein in which the fusion protein and the compound are cross-linked by an isopeptide bond. The fusion protein has a first peptide and protein containing a glutamine residue, and the compound has a lipid part or a fat-soluble vitamin part.

The method of reacting the fusion protein with the lipid peptide in the presence of the cell to which the lipidated protein is to be anchored is as follows. Compared with the method of tethering lipidated proteins to cell membranes, it is advantageous because operations such as purification are unnecessary.

[Lipidated protein]
One embodiment of a lipidated protein comprises a fusion protein portion having a first peptide and protein containing a glutamine residue, and a lipid portion or a fat-soluble vitamin portion. The lipid part or the fat-soluble vitamin part is bound to the fusion protein part by a glutamine residue of the first peptide and an isopeptide bond. The lipid part or the fat-soluble vitamin part may include a lipid peptide part having a second peptide containing a lysine residue.

Lipidated protein can also be obtained by the above-described method for producing lipidated protein. Therefore, the description content about the manufacturing method of the above-mentioned lipidated protein is applicable to lipidated protein. Conversely, the description of the lipidated protein of the present embodiment can be applied to the above-described method for producing a lipidated protein.

Lipidated proteins can interact with cell membranes such as anchoring to cell membranes by introducing lipids or fat-soluble vitamins. The lipidated protein according to the present embodiment can select various lipids that can be introduced into the protein, and by selecting the first peptide and the second peptide, and the first linker and the second linker, It is possible to control the degree of tethering to the cell membrane. The lipidated protein according to the present embodiment is useful for, for example, drugs, analytical reagents, enzyme reactions, and the like.

The 50% effective concentration (CC50) in the cell viability when the lipidated protein coexists with or acts on cells may be, for example, 1 μM or more, 5 μM or more, 10 μM or more, or 20 μM or more. The 50% effective concentration in cell viability when the lipidated protein coexists with or acts on cells may be, for example, 150 μM or less, or 140 μM or less. When the 50% effective concentration in the cell viability is within the above range, the safety of the lipidated protein can be further improved. The 50% effective concentration in cell viability can be adjusted, for example, by selecting the type of lipid part or fat-soluble vitamin part, the chain length of the lipid part, and the like. The 50% effective concentration in cell viability can be assessed by WST assay. Specifically, the 50% effective concentration in cell viability can be determined based on the method described in Examples described later.

As mentioned above, although embodiment of this invention was described, this invention is not limited to the said embodiment at all.

Hereinafter, the contents of the present invention will be described in more detail with reference to examples, comparative examples, and reference examples. However, the present invention is not limited to the following examples.

(Example 1)
[Preparation of fusion protein]
Based on the base sequence and amino acid sequence of highly sensitive green fluorescent protein (EGFP) (GeneBank accession number: HM640279, GI: 31205490), a fusion protein consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 3 was designed. Both of these fusion proteins have a first peptide portion containing a glutamine (Q) residue. In the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, EGFP and the first peptide are linked by a linker consisting of 4 bases of Gly-Gly-Gly-Ser. On the other hand, in the amino acid sequence shown in SEQ ID NO: 3, EGFP and the first peptide are directly bound.

Next, a nucleic acid encoding the designed fusion protein was synthesized. The nucleic acid was cloned into a cloning vector (pET22b +). Thereafter, the nucleic acid was genetically recombined by the PCR method to obtain an expression vector in which the target tag sequence was inserted. The sequence of this expression vector was identified by DNA sequencing.

The E. coli BL21 (DE3) strain was transformed with the above expression vector containing a nucleic acid encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 using the heat shock method. The transformed Escherichia coli was inoculated into an LB agar medium containing 100 μg / mL of ampicillin sodium, and allowed to stand at 37 ° C. overnight to obtain colonies. The obtained Escherichia coli colony was inoculated into 10 mL of LB medium containing 100 μg / mL of ampicillin sodium and cultured at 37 ° C. and 200 rpm for 4 hours. Thereafter, the cells are inoculated into 500 mL of LB medium and cultured at 37 ° C. and 120 rpm. When OD600 reaches 0.6, isopropyl-β-thiogalactopyranoside (Isopropyl β-D-1-Thiogalactopyranoside) is obtained. : IPTG) was added to a final concentration of 0.5 mM, the culture temperature was lowered to 15 ° C., and the culture was further continued for 16 hours.

After culturing, the culture was centrifuged at 6000 × g for 7 minutes to recover the cells. The obtained bacterial cells were washed three times with TBS buffer (mixture of 25 mM Tris-HCl and 150 mM NaCl aqueous solution, pH 7.4), the supernatant was removed, and the pelleted bacterial cells were- It was stored frozen at 80 ° C. After the cryopreserved pellet was dissolved in 15 mL of TBS buffer, the cells were pulverized by sonication and disrupted by centrifugation (temperature: 4 ° C., centrifugal force: 18000 × g, time: 20 minutes). Were separated from the protein-dissolved solution. The obtained solution was filtered through a 0.45 μm PVDF membrane filter and a 0.22 μm PVDF membrane filter to remove insoluble fractions and bacterial cells. Protein was purified using a HisTrap Excel column (1 mL) with respect to the solution from which the insoluble fraction and the like were removed. SDS-PAGE was performed on the obtained protein, and the appearance of a band corresponding to the molecular weight band corresponding to the designed fusion protein was observed, confirming the expression and purity of the fusion protein.

[Preparation of compound having lipid part (lipid peptide)]
The Fmoc solid phase synthesis method is used to prepare a second peptide by condensing the amino acid sequences shown in Table 1 below, one by one, and then the amino group and myristic acid of the N-terminal amino acid of the second peptide are prepared. Lipid peptide 1 was synthesized by condensation. The resulting lipid peptide was purified by reverse phase liquid chromatography (RP-HPLC). Identification of the lipid peptide after purification and measurement of purity were performed by MALDI-TOF-MS and RP-HPLC. In addition, lipid peptides in which myristic acid and the second peptide were connected by a linker were synthesized in the same manner (lipid peptides 2 to 12 in Table 1). Lipid peptides were synthesized in the same manner using lauric acid, palmitic acid, and stearic acid instead of myristic acid (lipid peptides 13 to 15 in Table 1). Lipid peptides were synthesized in the same manner using a peptide shorter than MRHKGS as the second peptide (lipid peptides 16 to 21 in Table 1). A lipid peptide was synthesized using lithocholic acid instead of myristic acid (lipid peptide 22 in Table 1).

Measurement using RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 250 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The elution of lipid peptide (0.5 mg / mL, 20 μL) was performed under the condition that the mobile phase was fed at a flow rate of 1.0 mL / min so that the concentration gradient of acetonitrile would be 30% to 60% after 30 minutes. The absorbance was detected. Measurement using MALDI-TOF-MS was performed using 2 μL of a 0.5 mg / mL lipid peptide solution and an α-cyano-4-hydroxycinnamic acid (CHCA) matrix.

[Cytotoxicity test of lipid peptides]
Toxicity of synthesized lipid peptides to cells was evaluated by WST assay. Floating cells SNU-1 washed twice with 100 μL phosphate buffered saline (PBS) 5.0 × 10 ⁴ cells / well (25 μL liquid medium (Opti-MEM, Life Technologies Japan)) and Inoculated into a 96-well plate. Then, 25 μL of the lipid peptide synthesized above (0 to 1000 μM) was added and mixed to prepare a solution with a final concentration of 0 to 500 μM. The obtained solution was incubated at 37 ° C. for one day. After the incubation, a color former (WST-8, manufactured by Dojindo Laboratories) diluted 6-fold with Opti-MEM was added to 60 μL / well and incubated at 37 ° C. for 90 minutes. Then, the absorbance at 450 nm was measured, and the cell viability was calculated. Cell viability was calculated from the following formula. The results are shown in FIG. From FIG. 1, CC50, which is an index of cytotoxicity, was calculated from a sigmoidal regression curve of KaleidaGraph. The results are shown in Table 2.

Cell viability [%] = [OD450 (sample) −OD450 (blank)] / [OD450 (untreated) −OD450 (blank)] × 100
In the above formula, OD represents optical density.

[Measurement of critical micelle concentration (CMC) of lipid peptide]
The critical micelle concentration of the lipid peptides shown in Table 2 was measured. The lipid peptide concentration was measured according to the pyrene method (J. Aguar et al., J. Colloid Interface Sci. 2003, 258, p. 116-122). First, 100 μM pyrene (tetrahydrofuran solution) was taken in a 2 μL microtube, and tetrahydrofuran was evaporated. Thereafter, 100 μL of lipid peptide (0.0001 to 0.5 mM, PBS solution) was added to the microtube and incubated at 25 ° C. and 15 rpm for 60 minutes. After incubation, a PBS solution was added to a 96-well plate at 50 μL / well, and the ratio of fluorescence intensity at ₃₈₅ nm (I ₃₈₅ ) to fluorescence intensity at ₃₇₃ nm (I ₃₇₃ ) (I ₃₈₅ / I ₃₇₃ ) was measured. The wavelength of the excitation light was set at 334 nm. The results when lipid peptide 1 is used are shown in FIG. The critical micelle concentration was determined from the change in the I ₃₈₅ / I ₃₇₃ ratio in FIG. The results are shown in Table 2.

1. From the results shown in FIG. 1, it was confirmed that any lipid peptide had a large CC50 value, and the cytotoxicity was low when the lipid peptide was used at a concentration of about 1 μM. It was also confirmed that cytotoxicity can be adjusted by selecting a second linker. From the results shown in Table 2, it was confirmed that the critical micelle concentration can be adjusted by selecting the second linker.

[Measurement of circular dichroism spectra of lipid peptides]
The circular dichroism spectrum of the synthesized lipid peptide was measured and the secondary structure was evaluated. First, 50 μM lipid peptide was dissolved in 400 μL of PBS and incubated at 37 ° C. for 30 minutes. After the incubation, the circular dichroism spectrum of the PBS solution was measured using a quartz cell having an optical path length of 0.1 cm. The measurement was performed at a temperature of 37 ° C., a scanning speed of 50 nm / min, and a data acquisition interval of 0.2 nm. The results are shown in FIGS.

3 and 4 are graphs showing the results of circular dichroism spectrum measurement of the synthesized lipid peptide. FIG. 3 shows the results for

lipid peptides

1, 3, 6, and 9-12, and FIG. 4 shows the results for lipid peptides 9, and 16-21. From the results shown in FIG. 3, lipid peptides 9 to 11 have a negative peak at 215 to 225 nm, confirming that the lipid peptide forms a β sheet structure. Moreover, since the lipid peptides 6 and 12 have a negative peak at 205 nm, it was confirmed that the lipid peptides formed a random coil structure. From the results shown in FIG. 4, it was confirmed that the

lipid peptides

16, 17 and 20 also formed a β sheet structure like the lipid peptide 9. It was also confirmed that lipid peptides 19 and 21 formed a random coil structure. By using a lipid peptide that forms a β-sheet structure, it is expected to improve the anchoring ability to the cell membrane.

[Reaction between fusion protein and lipid peptide]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1, 50 μM of the lipid peptide 1, and 0.1 U / mL of microorganism-derived transglutaminase were mixed in 10 mL of PBS, and the conditions were 37 ° C. And reacted for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 5. After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained. The results are shown in FIG.

The chromatogram shown in the upper part of FIG. 5 shows the result of liquid chromatography on the solution before the reaction. It was confirmed that an elution peak corresponding to the fusion protein was observed after 13.4 minutes. Further, the chromatogram shown at the bottom of FIG. 5 shows the result of liquid chromatography on the solution after the reaction. In addition to the elution peak corresponding to the fusion protein being observed after 13.4 minutes, a new elution peak corresponding to the lipidated protein was observed after 14.3 minutes. From the results shown in FIG. 5, it was confirmed that the transglutaminase reaction was progressing. Further, the reaction rate in the transglutaminase reaction was calculated from the peak area of the chromatogram shown in FIG.

Reaction rate in transglutaminase reaction [%] = [(peak area with respect to peak after 14.3 minutes) / {(peak area with respect to peak after 13.4 minutes) + (peak area with respect to peak after 14.3 minutes)] }] × 100

Measurement by RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 150 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The elution of the lipidated protein was carried out under the condition that the mobile phase was fed at a flow rate of 1 mL / min so that the concentration gradient of acetonitrile would be 20% to 100% after 40 minutes, and the absorbance was detected at 280 nm.

When the K / Q ratio was 5 and the reaction time was 30 minutes, the reactivity of the transglutaminase reaction of the fusion protein and lipid peptide was evaluated according to the following criteria. The results are shown in Table 3.
<Reactivity evaluation criteria>
The reactivity was evaluated based on the ratio of lipid peptide introduced to the fusion protein required for the reaction.
A: 100% by mass of fusion protein reacted with lipid peptide
B: Fusion protein reacted with lipid peptide exceeds 60% by mass and less than 100% C: Fusion protein reacted with lipid peptide is less than 60% by mass

Next, the K / Q ratio was changed to 1, 2, 10, and 20, and the same experiment as described above was performed to calculate the reaction rate at each K / Q ratio. The results are shown in FIG. In addition, sampling was performed every 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes, and the reaction rate after each reaction time was calculated. The results are shown in FIG.

(Example 2)
The reaction rate was calculated in the same manner as in Example 1 except that lipid peptide 9 having a linker was used instead of lipid peptide 1. The results are shown in Table 3, FIG. 6 and FIG.

(Example 3)
An experiment was conducted in the same manner as in Example 1 except that the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 was used instead of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1. The reaction rate was calculated. The results are shown in Table 3, FIG. 8 and FIG.

Example 4
An experiment was conducted in the same manner as in Example 2 except that the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 was used instead of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1. The reaction rate was calculated. The results are shown in Table 3, FIG. 8 and FIG.

(Example 5)
An experiment was conducted in the same manner as in Example 1 except that the fusion protein having the amino acid sequence represented by SEQ ID NO: 3 was used instead of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1. . Regarding the evaluation, the relationship between the K / Q ratio and the reaction rate was evaluated. The results are shown in Table 3 and FIG.

(Example 6)
An experiment was conducted in the same manner as in Example 2 except that the fusion protein having the amino acid sequence represented by SEQ ID NO: 3 was used instead of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1. The reaction rate was calculated. The results are shown in Table 3, FIG. 10 and FIG.

From the results shown in FIG. 6 to FIG. 11, it was confirmed that lipidated protein can be produced in any relationship between the fusion protein and the lipid peptide. When using a fusion protein having the amino acid sequence represented by SEQ ID NO: 1, it was confirmed that the reactivity was excellent even when the K / Q ratio was small, regardless of the type of lipid peptide. Moreover, when using the fusion protein which has an amino acid sequence represented by sequence number 2, it was confirmed that lipid can be introduce | transduced into all the fusion proteins by adjusting K / Q ratio.

(Example 7)
[Reaction between fusion protein and lipid peptide]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 above, 50 μM of the lipid peptide 6 and 0.1 U / mL of the microorganism-derived transglutaminase were mixed in 10 mL of PBS, under the conditions of 37 ° C. And reacted for 60 minutes. At this time, the K / Q ratio was set to 5. After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained.

Next, the K / Q ratio was changed to 1, 2, 10, and 20, and the same experiment as described above was performed to evaluate the reactivity. The results are shown in FIG. In addition, sampling was performed every 5 minutes, 10 minutes, 15 minutes, 30 minutes, and 60 minutes to evaluate the reactivity. The results are shown in Table 3 and FIG. 12 and 13 also show the examples manufactured in Example 3 and Example 4 for comparison.

(Examples 8 to 10)
Instead of using lipid peptide 6 in Table 1 (Example 8), lipid peptide 11 in Table 1 (Example 9), or lipid peptide 12 in Table 1 (Example 10) instead of lipid peptide 6, Experiments and evaluations were performed in the same manner as in Example 7. The results are shown in Table 3, FIG. 12 and FIG.

(Examples 11 to 16)
Instead of lipid peptide 6, lipid peptide 16 in Table 1 (Example 11), lipid peptide 17 in Table 1 (Example 12), lipid peptide 18 in Table 1 (Example 13), lipid peptide 19 in Table 1 (Example 13) Example 14), lipid peptide 20 of Table 1 (Example 15), or lipid peptide 21 of Table 1 (Example 16) was used except that the experiment was conducted and evaluated in the same manner as in Example 7. It was. The results are shown in Table 3, FIG. 14 and FIG.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the reaction solutions of lipidated proteins (including unreacted lipid peptides) obtained in Example 3, Example 4, and Examples 8 to 10, the ability of the lipidated proteins to anchor to the cell membrane was evaluated. did. First, 2.0 × 10 ⁵ floating cells SNU-1 were seeded, washed twice with 200 μL of PBS, and then suspended in 25 μL of PBS. The resulting suspension of suspension cells was mixed with 25 μL of the lipidated protein (2 μM or 10 μM) obtained above to prepare a solution with a final concentration of 1 μM or 5 μM. The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG.

Based on the quantified amount of fluorescence, the ability of the lipidated protein to anchor to the cell membrane was evaluated according to the following criteria. The results are shown in Table 3.
<Evaluation criteria for mooring ability (fluorescence intensity per cell)>
A: 1000 or more B: 500 or more and less than 1000 C: 200 or more and less than 500 D: 150 or more and less than 200 E: 100 or more and less than 150 F: 50 or more and less than 100 G: less than 50

(Reference Example 1)
As a reference example, the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 before reacting with the lipid peptide was directly added to the suspension of suspension cells to prepare a solution having a final concentration of 1 μM or 5 μM. . The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in Table 3 and FIG.

[Cytotoxicity test]
Using the lipidated protein reaction solutions (including unreacted lipid peptides) obtained in Example 3, Example 4, and Examples 8 to 10, cytotoxicity of the lipidated protein was evaluated by WST assay. did. First, floating cells SNU-1 washed twice with 100 μL of PBS was seeded in a 96-well plate at 5.0 × 10 ⁴ cells / well (25 μL Dulbecco's PBS (D-PBS)). Then, 25 μL of the above reaction solution (2 μM or 10 μM, the concentration of which was adjusted with D-PBS if necessary) was mixed to prepare a solution with a final concentration of 1 μM or 2 μM. The resulting solution was incubated at 37 ° C. for 15 minutes. After incubation, the plate was washed twice with 100 μL of D-PBS. WST-8 diluted with Opti-MEM was added at 110 μL / well and incubated at 37 ° C. for 90 minutes. Thereafter, the absorbance at 450 nm was measured to calculate the cell viability. The results are shown in FIG.

(Reference Example 2)
As a reference example, the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 before reacting with the lipid peptide was directly added to the suspension of suspension cells to prepare a solution having a final concentration of 1 μM or 5 μM. . The resulting solution was incubated at 37 ° C. for 15 minutes. After incubation, the plate was washed twice with 100 μL of D-PBS. 110 μL / well of WST-8 diluted with Opti-MEM was added and incubated for 90 minutes. Thereafter, the absorbance at 450 nm was measured to calculate the cell viability. The results are shown in FIG.

From the results shown in FIG. 17, even when the lipidated protein is used at a concentration of about 1 μM, the cell viability of the cells to which the lipidated protein is anchored is close to 100% and is sufficiently biocompatible. That was confirmed.

Using the reaction solution (including unreacted lipid peptide) of the lipidated protein obtained in Example 4 and Examples 11 to 16, the anchoring behavior and cytotoxicity of the lipidated protein to the cell membrane were evaluated. The results are shown in Table 3, FIG. 18 and FIG.

From the results shown in FIG. 19, even when the lipidated protein is used at a concentration of about 1 μM, the cell viability of the cells to which the lipidated protein is anchored is close to 100%, and is sufficiently biocompatible. That was confirmed.

(Examples 17 to 20)
Implemented except that lipid peptide 3 (Example 17), lipid peptide 4 (Example 18), lipid peptide 8 (Example 19) or lipid peptide 7 (Example 20) was used instead of lipid peptide 1. Experiments were conducted in the same manner as in Example 7 to confirm that lipidated proteins can be produced. The results are shown in Table 3.

(Example 21)
[Preparation of compound having fat-soluble vitamin part]
By Fmoc solid phase synthesis, the amino acid sequence represented by GGGSMRHKGS is synthesized by condensing one amino acid at a time, and then the amino group of the N-terminal amino acid and α-tocopherol are condensed to synthesize a compound having a fat-soluble vitamin part. did. The obtained compound having a fat-soluble vitamin part was purified by reverse phase liquid chromatography (RP-HPLC). Identification of the lipid peptide after purification and measurement of purity were performed by MALDI-TOF-MS and RP-HPLC.

Measurement using RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 250 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. For elution of the compound having a fat-soluble vitamin part (0.5 mg / mL, 20 μL), the mobile phase is fed at a flow rate of 1.0 mL / min so that the concentration gradient of acetonitrile becomes 50% to 80% after 30 minutes. The detection was carried out under the conditions, and the absorbance was 220 nm. The measurement using MALDI-TOF-MS was performed using 2 μL of a solution of a compound having a fat-soluble vitamin part of 0.5 mg / mL and an α-cyano-4-hydroxycinnamic acid (CHCA) matrix. It was.

[Reaction between fusion protein and compound with fat-soluble vitamin]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2, 50 μM of the compound having the fat-soluble vitamin part, 0.1 U / mL of microorganism-derived transglutaminase, 10 mM Tris-HCl (pH 8.0) ) And 1% by mass / volume in a mixed solution of n-dodecyl-β-D-maltoside (DDM) and allowed to react at 37 ° C. for 60 minutes. It was. At this time, the molar ratio (K) of the primary amino group (here, lysine (K) residue) in the compound having a fat-soluble vitamin part and the glutamine (Q) residue contained in the first peptide in the fusion protein. / Q ratio) was set to 5. After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein (purity: 99%) was obtained.

(Example 22)
[Reaction between fusion protein and compound having lipid part (lipid peptide)]
A lipidated protein was prepared in the same manner as in Example 21 except that the lipid peptide 22 shown in Table 1 was used instead of the compound having the fat-soluble vitamin part. It was confirmed by RP-HPLC that lipidated protein (purity: 80%) was obtained.

(Example 23)
[Reaction between fusion protein and compound having lipid part]
A lipidated protein was produced in the same manner as in Example 21 except that tetradecylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of the compound having a fat-soluble vitamin part. That is, 10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 above, 50 μM of tetradecylamine, and 0.1 U / mL of microorganism-derived transglutaminase were mixed in 10 mL of PBS, and the conditions at 37 ° C. The reaction was allowed to proceed for 30 minutes. At this time, the molar ratio (K / Q ratio) between the primary amino group contained in the compound having a lipid part and the glutamine (Q) residue contained in the first peptide in the fusion protein was set to 5. After 30 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained. Tetradecylamine was a 5 mM solution prepared by dissolving in dimethyl sulfoxide.

(Example 24)
An experiment was performed in the same manner as in Example 23 except that hexadecylamine (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of tetradecylamine, and it was confirmed that a lipidated protein was obtained.

(Example 25)
[Reaction between fusion protein and lipid peptide]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 1, 50 μM of lipid peptide 9 of Table 1, and 0.1 U / mL of microorganism-derived transglutaminase in 10 mM Tris-HCl (pH 8.0) And reacted at 37 ° C. for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 1, 5 or 20. After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein (purity: 91%) was obtained. The result of the reaction rate in each K / Q ratio is shown in FIG.

(Examples 26 to 28)
The procedure was carried out except that lipid peptide 13 in Table 1 (Example 26), lipid peptide 14 in Table 1 (Example 27) or lipid peptide 15 in Table 1 (Example 28) was used in place of lipid peptide 9. In the same manner as in Example 25, lipidated protein was produced. The purity of the lipidated protein was 48% in Example 26, 96% in Example 27, and 90% in Example 28, respectively. The result of the reaction rate in each K / Q ratio is shown in FIG.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the lipidated protein reaction solutions obtained in Examples 21 to 22 and 25 to 28 (including compounds having unreacted fat-soluble vitamins), the ability of the lipidated protein to anchor to the cell membrane was evaluated. The evaluation method was performed in the same manner as in Example 3 above. The results are shown in FIG. The fluorescence microscope observation results of Examples 25 to 28 are shown in FIGS.

[Cytotoxicity test]
Using the reaction solutions of lipidated proteins obtained in Examples 21 and 22 and Examples 26 to 28 (including compounds having unreacted fat-soluble vitamins), cytotoxicity of lipidated proteins was evaluated by WST assay. did. The evaluation method was performed in the same manner as in Example 3 above. The results are shown in FIGS.

[Measurement of cytotoxicity and critical micelle concentration of compounds having lipid part or fat-soluble vitamin part]
The cytotoxicity and critical micelle concentration of the compounds having lipid part or fat-soluble vitamin part used in Examples 21 to 22 and Examples 25 to 28 were measured. The results are shown in Table 4.

(Example 29)
The Fmoc solid phase synthesis method was used to prepare a second peptide by condensing the amino acid sequences shown in Table 5 below, one by one, and then preparing the amino group of the N-terminal amino acid of the second peptide and α-tocopherol Were connected by a second linker to synthesize lipid peptide 23. The resulting lipid peptide was purified by RP-HPLC. Identification of the lipid peptide after purification and measurement of purity were performed by MALDI-TOF-MS and RP-HPLC. Lipid peptides were synthesized in the same manner using cholesterol instead of α-tocopherol (lipid peptide 24 in Table 5). The lipid peptide 24 is represented by the following chemical formula.

Measurement using RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 250 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The elution of lipid peptide (0.5 mg / mL, 20 μL) was carried out under the condition that the mobile phase was fed at a flow rate of 1.0 mL / min so that the concentration gradient of acetonitrile would be 30% or 60% after 30 minutes. The absorbance was detected. Measurement using MALDI-TOF-MS was performed using 2 μL of a 0.5 mg / mL lipid peptide solution and an α-cyano-4-hydroxycinnamic acid (CHCA) matrix.

For the lipid peptide 23 and lipid peptide 24 synthesized as described above, the cytotoxicity test and the critical micelle concentration were evaluated in the same manner as in Example 1. The results are shown in Table 6. The measurement results when lipid peptide 24 is used are shown in FIG. For comparison, the evaluation results of lipid peptide 9, lipid peptide 13, lipid peptide 14, lipid peptide 15, and lipid peptide 22 synthesized in Example 1 are also shown in Table 6.

[Reaction between fusion protein and lipid peptide]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2, 50 μM of the lipid peptide 13, and 0.1 U / mL of the microorganism-derived transglutaminase were mixed in 10 mM Tris-HCl (pH 8.0). And allowed to react at 37 ° C. for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 5.

After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained. The reactivity was evaluated in the same manner as in Example 1. The results are shown in Table 7.

Measurement using RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 250 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The elution of lipid peptide (0.5 mg / mL, 20 μL) was carried out under the condition that the mobile phase was fed at a flow rate of 1.0 mL / min so that the concentration gradient of acetonitrile would be 20% or 100% after 40 minutes. The absorbance was detected.

(Examples 30 to 34)
As described in Table 7, instead of lipid peptide 13, lipid peptide 14 (Example 30), lipid peptide 15 (Example 31), lipid peptide 22 (Example 32), lipid peptide 23 (Example 33), In addition, the experiment was conducted in the same manner as in Example 29 except that lipid peptide 24 (Example 34) was used, and the reactivity of the transglutaminase reaction of the fusion protein and the lipid peptide was evaluated. The results are shown in Table 7.

(Example 35)
As shown in Table 7, the experiment was carried out in the same manner as in Example 4 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 36)
As shown in Table 7, the experiment was performed in the same manner as in Example 29 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 37)
As shown in Table 7, the experiment was performed in the same manner as in Example 30 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 38)
As shown in Table 7, the experiment was carried out in the same manner as in Example 31 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 39)
As shown in Table 7, the experiment was conducted in the same manner as in Example 32 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 40)
As shown in Table 7, the experiment was performed in the same manner as in Example 33 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

(Example 41)
As shown in Table 7, the experiment was performed in the same manner as in Example 34 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 7.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the lipidated protein reaction solutions (including unreacted lipid peptide) obtained in Examples 29 to 41, the ability of the lipidated protein to anchor to the cell membrane was evaluated. First, 2.0 × 10 ⁵ floating cells SNU-1 were seeded, washed twice with 100 μL of PBS, and then suspended in 25 μL of PBS. The resulting suspension of suspension cells was mixed with 25 μL of the lipidated protein (2 μM) obtained above to prepare a solution with a final concentration of 1 μM. The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG.

(Reference Example 3)
As a reference example, the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 before reacting with the lipid peptide was directly added to the suspension of suspension cells to prepare a solution having a final concentration of 1 μM or 5 μM. . The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG.

When a lipid peptide in which a fatty acid is bonded to the N-terminus of a peptide having a glutamine residue is used as a substrate as in a conventional method for producing a lipidated protein using transglutaminase, the carbon number of the lipid part is relatively small. When it is large (for example, having 16 or more carbon atoms), the water solubility is low, and it was difficult to carry out the transglutaminase reaction without the addition of a surfactant. However, from the results in Table 7, even when lipid peptide 14, lipid peptide 15, lipid peptide 23, and lipid peptide 24 having a relatively large number of carbon atoms are used, the transglutaminase reaction proceeds and lipidated proteins are It was confirmed that it could be manufactured. Further, from the results shown in Table 7, the reactivity of the transglutaminase reaction could be further improved in the examples using the surfactant (Examples 35 to 41).

In addition, when a lipid peptide in which a fatty acid is bonded to the N-terminus of a peptide having a glutamine residue is used as a substrate as in a conventional method for producing a lipidated protein using transglutaminase, the number of carbons in the lipid portion increases. However, since purification of the lipid peptide itself becomes difficult, the choice of lipid peptide for producing a lipidated protein tends to be limited. As described above, according to the method of the present disclosure, it has been confirmed that the choice of lipid peptides can be expanded more than before.

[Cytotoxicity test]
Using the lipidated protein reaction solutions obtained in Examples 29 to 34 (including compounds having unreacted fat-soluble vitamins), the cytotoxicity of lipidated proteins was evaluated by WST assay. The evaluation method was performed in the same manner as in Example 3 above. The results are shown in FIG.

(Reference Example 4)
As a reference example, the fusion protein having the amino acid sequence represented by SEQ ID NO: 2 before reacting with the lipid peptide was directly added to the suspension of suspension cells to prepare a solution having a final concentration of 1 μM or 5 μM. . The resulting solution was incubated at 37 ° C. for 15 minutes. After incubation, the plate was washed twice with 100 μL of D-PBS. 110 μL / well of WST-8 diluted with Opti-MEM was added and incubated for 90 minutes. Thereafter, the absorbance at 450 nm was measured to calculate the cell viability. The results are shown in FIG.

(Example 42)
The Fmoc solid phase synthesis method was used to prepare a second peptide by condensing the amino acid sequences listed in Table 8 below, one amino acid at a time, and then the amino group of the N-terminal amino acid of the second peptide, lauric acid, Were connected by a second linker to synthesize lipid peptide 25. The resulting lipid peptide was purified by RP-HPLC. Identification of the lipid peptide after purification and measurement of purity were performed by MALDI-TOF-MS and RP-HPLC. Further, lipid peptides were synthesized in the same manner using palmitic acid, stearic acid, oleic acid, linoleic acid or cholesterol instead of lauric acid (lipid peptides 26 to 30 in Table 8).

Cytotoxicity tests and critical micelle concentrations were evaluated for lipid peptides 25 to 30 synthesized as described above.

[Cytotoxicity test of lipid peptides]
Toxicity of synthesized lipid peptides to cells was evaluated by WST assay. 1.0 × 10 ⁴ floating cells SNU-1 washed twice with 100 μL phosphate buffered saline (PBS) (25 μL liquid medium (Opti-MEM, Life Technologies Japan)) and Inoculated into a 96-well plate. Then, 25 μL of the lipid peptide synthesized above (0 to 1000 μM) was added and mixed to prepare a solution with a final concentration of 0 to 500 μM. The obtained solution was incubated at 37 ° C. for one day. After the incubation, a color former (WST-8, manufactured by Dojindo Laboratories) diluted 6-fold with Opti-MEM was added to 60 μL / well and incubated at 37 ° C. for 90 minutes. Then, the absorbance at 450 nm was measured, and the cell viability was calculated. Cell viability was calculated using the same formula as shown in Example 1. The results are shown in FIG. 29 and FIG. From FIG. 29 and FIG. 30, CC50, which is an index of cytotoxicity, was calculated using a sigmoid regression curve of KaleidaGraph. The results are shown in Table 9. For comparison, the results of lipid peptide 16 synthesized in Example 1 are also shown in Table 9.

[Measurement of critical micelle concentration (CMC) of lipid peptide]
The critical micelle concentration of lipid peptide 25 to lipid peptide 30 was measured. The lipid peptide concentration was measured according to the pyrene method (J. Aguar et al., J. Colloid Interface Sci. 2003, 258, p. 116-122). First, 100 μM pyrene (tetrahydrofuran solution) was taken in a 2 μL microtube, and tetrahydrofuran was evaporated. Thereafter, 100 μL of lipid peptide (0.0001 to 0.5 mM, PBS solution) was added to the microtube and incubated at 25 ° C. and 15 rpm for 60 minutes. After incubation, a PBS solution was added to a 96-well plate at 50 μL / well, and the ratio of fluorescence intensity at ₃₈₅ nm (I ₃₈₅ ) to fluorescence intensity at ₃₇₃ nm (I ₃₇₃ ) (I ₃₈₅ / I ₃₇₃ ) was measured. The wavelength of the excitation light was set at 334 nm. Similarly to Example 1, a change in the I ₃₈₅ / I ₃₇₃ ratio was _derived from the graph showing the results of fluorescence intensity measurement when lipid peptides were used, and the critical micelle concentration was determined. The results are shown in Table 9. For comparison, the results of lipid peptide 16 synthesized in Example 1 are also shown in Table 9.

[Reaction between fusion protein and lipid peptide]
10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2, 50 μM of the lipid peptide 25, and 0.1 U / mL of the microorganism-derived transglutaminase were mixed in 10 mM Tris-HCl (pH 8.0). And allowed to react at 37 ° C. for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 5.

After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained. The reactivity was evaluated in the same manner as in Example 1. The results are shown in Table 10. For comparison, the results of Example 11 are also shown in Table 9.

Measurement using RP-HPLC was performed at room temperature (25 ° C.) using a 4.6 × 250 mm Inertsil ODS-3 column. The mobile phase used was Milli-Q water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The elution of lipid peptide (0.5 mg / mL, 20 μL) was performed under the condition that the mobile phase was fed at a flow rate of 1.0 mL / min so that the concentration gradient of acetonitrile would be 20% to 100% after 40 minutes. The absorbance was detected.

(Examples 43 to 47)
As shown in Table 10, instead of lipid peptide 25, lipid peptide 26 (Example 43), lipid peptide 27 (Example 44), lipid peptide 28 (Example 45), lipid peptide 29 (Example 46), In addition, experiments were conducted in the same manner as in Example 42 except that lipid peptide 30 (Example 47) was used, and the reactivity of the transglutaminase reaction of the fusion protein and the lipid peptide was evaluated. The results are shown in Table 10.

(Example 48)
As shown in Table 10, the experiment was conducted in the same manner as in Example 11 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 49)
As shown in Table 10, the experiment was conducted in the same manner as in Example 42 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 50)
As shown in Table 10, the experiment was conducted in the same manner as in Example 43 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 51)
As shown in Table 10, the experiment was performed in the same manner as in Example 44 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 52)
As shown in Table 10, the experiment was conducted in the same manner as in Example 45 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 53)
As shown in Table 10, the experiment was conducted in the same manner as in Example 46 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

(Example 54)
As shown in Table 10, the experiment was conducted in the same manner as in Example 47 except that n-dodecyl-β-D-maltoside was added as a surfactant, and the reaction of the transglutaminase reaction of the fusion protein and lipid peptide was performed. Sex was evaluated. The results are shown in Table 10.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the lipidated protein reaction solutions (including unreacted lipid peptides) obtained in Example 11 and Examples 42 to 54, the ability of the lipidated protein to anchor to the cell membrane was evaluated. First, 2.0 × 10 ⁵ floating cells SNU-1 were seeded, washed twice with 100 μL of PBS, and then suspended in 25 μL of PBS. The resulting suspension of suspension cells was mixed with 25 μL of the lipidated protein (2 μM) obtained above to prepare a solution with a final concentration of 1 μM. The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG. 31 and Table 10.

[Cytotoxicity test]
Using the lipidated protein reaction solutions obtained in Examples 42 to 47 (including compounds having unreacted fat-soluble vitamins), the cytotoxicity of lipidated proteins was evaluated by WST assay. The evaluation method was performed in the same manner as in Example 3 above. The results are shown in FIG.

(Example 55)
The Fmoc solid phase synthesis method was used to prepare a second peptide by condensing the amino acid sequences shown in Table 11 below one by one, and then preparing the second peptide with the amino group of the N-terminal amino acid, lauric acid, Were synthesized with a lipid peptide 31 connected by a linker. The resulting lipid peptide was purified by RP-HPLC. Identification of the lipid peptide after purification and measurement of purity were performed by MALDI-TOF-MS and RP-HPLC. Lipid peptides were synthesized in the same manner using palmitic acid, stearic acid, α-tocopherol or cholesterol instead of lauric acid (lipid peptides 32-35 in Table 11).

In addition, as a second linker, by introducing lysine (K) into the lipid part side of G ₃ S, a lipid peptide into which two lipid parts have been introduced is also synthesized. The measurement was performed by MALDI-TOF-MS and RP-HPLC. More specifically, lipid peptide 36 introduced with two lauric acids, lipid peptide 37 introduced with two myristic acids, lipid peptide 38 introduced with two palmitic acids, lipid peptide 39 introduced with two stearic acids, Lipid peptide 40 introduced with two α-tocopherols and lipid peptide 41 introduced with two cholesterols were synthesized. The lipid peptide 36 is represented by the following chemical formula.

For the lipid peptides 31 to 41 synthesized as described above, cytotoxicity tests and critical micelle concentrations were evaluated.

[Cytotoxicity test of lipid peptides]
Toxicity of synthesized lipid peptides to cells was evaluated by WST assay. 1.0 × 10 ⁴ floating cells SNU-1 washed twice with 100 μL phosphate buffered saline (PBS) (25 μL liquid medium (Opti-MEM, Life Technologies Japan)) and Inoculated into a 96-well plate. Then, 25 μL of the lipid peptide synthesized above (0 to 1000 μM) was added and mixed to prepare a solution with a final concentration of 0 to 500 μM. The obtained solution was incubated at 37 ° C. for one day. After the incubation, a color former (WST-8, manufactured by Dojindo Laboratories) diluted 6-fold with Opti-MEM was added to 60 μL / well and incubated at 37 ° C. for 90 minutes. Then, the absorbance at 450 nm was measured, and the cell viability was calculated. Cell viability was calculated using the same formula as shown in Example 1. The results are shown in FIG. 33, FIG. 34 and FIG. From FIG. 33, FIG. 34 and FIG. 35, CC50 as an index of cytotoxicity was calculated using a sigmoid regression curve of KaleidaGraph. The results are shown in Table 12. For comparison, the results of the lipid peptide 21 synthesized in Example 1 are also shown in Table 12.

[Measurement of critical micelle concentration (CMC) of lipid peptide]
The critical micelle concentrations of lipid peptide 21 and lipid peptide 31 to lipid peptide 41 were measured. The lipid peptide concentration was measured according to the pyrene method (J. Aguar et al., J. Colloid Interface Sci. 2003, 258, p. 116-122). First, 100 μM pyrene (tetrahydrofuran solution) was taken in a 2 μL microtube, and tetrahydrofuran was evaporated. Thereafter, 100 μL of lipid peptide (0.0001 to 0.5 mM, PBS solution) was added to the microtube and incubated at 25 ° C. and 15 rpm for 60 minutes. After incubation, a PBS solution was added to a 96-well plate at 50 μL / well, and the ratio of fluorescence intensity at ₃₈₅ nm (I ₃₈₅ ) to fluorescence intensity at ₃₇₃ nm (I ₃₇₃ ) (I ₃₈₅ / I ₃₇₃ ) was measured. The wavelength of the excitation light was set at 334 nm. Similarly to Example 1, a change in the I ₃₈₅ / I ₃₇₃ ratio was _derived from the graph showing the results of fluorescence intensity measurement when lipid peptides were used, and the critical micelle concentration was determined. The results are shown in Table 12.

[Reaction between fusion protein and lipid peptide]
The fusion protein having the amino acid sequence represented by SEQ ID NO: 2 is 10 μM, the lipid peptide 31 is 50 μM, the microorganism-derived transglutaminase is 0.1 U / mL, and n-dodecyl-β-D-maltoside is 1 as a surfactant. 0.0 mass / volume% was mixed in 10 mL of PBS and reacted at 37 ° C. for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. The (K / Q ratio) was 10. The same reaction as described above was performed by changing the K / Q ratio to 5 or 20. The results are shown in FIG.

After 60 minutes, 30 μL of 0.2% TFA-containing acetonitrile was added to stop the reaction. It was confirmed by RP-HPLC that lipidated protein was obtained. The reactivity was evaluated in the same manner as in Example 1. The results are shown in Table 13.

(Examples 56 to 66)
As described in Table 13, instead of lipid peptide 31, lipid peptide 32 (Example 56), lipid peptide 33 (Example 57), lipid peptide 34 (Example 58), lipid peptide 35 (Example 59), Lipid peptide 36 (Example 60), Lipid peptide 37 (Example 61), Lipid peptide 38 (Example 62), Lipid peptide 39 (Example 63), Lipid peptide 40 (Example 64), Lipid peptide 41 (Execute) Example 55), except that lipid peptide 21 (Example 66) was used, and that the amount of each lipid peptide was adjusted so that the K / Q ratio was 5, 10, 20 and Example 55 The experiment was conducted in the same manner, and the reactivity of the transglutaminase reaction of the fusion protein and lipid peptide was evaluated. The results are shown in FIGS. 36 and 37 and Table 13.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the lipidated protein reaction solutions (including unreacted lipid peptides) obtained in Examples 55 to 66, the ability of the lipidated protein to anchor to the cell membrane was evaluated. First, 2.0 × 10 ⁵ floating cells SNU-1 were seeded, washed twice with 100 μL of PBS, and then suspended in 25 μL of PBS. The resulting suspension of suspension cells was mixed with 25 μL of the lipidated protein (2 μM) obtained above to prepare a solution with a final concentration of 1 μM. The mixed solution was incubated at 37 ° C. for 15 minutes. After incubation, the solution was washed twice with 200 μL PBS. The washed solution was suspended in 400 μL of PBS and filtered with a cell strainer. Thereafter, the fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG. 38 and Table 13. For comparison, the results of Example 16 are also shown in Table 13.

The portion indicated by “- ^* ” in Table 13 means that no measurement is performed.

[Cytotoxicity test]
Cytotoxicity of lipidated proteins by WST assay using the reaction solutions of lipidated proteins obtained in Examples 55 to 57 and Examples 59 to 63 and 66 (containing compounds having unreacted fat-soluble vitamins) Evaluated. The evaluation method was performed in the same manner as in Example 3 above. The results are shown in FIG.

(Example 67)
[Reaction between fusion protein and lipid peptide]
The reaction between the fusion protein and the lipid peptide was carried out in the presence of the cells to be anchored with the lipidated protein.

10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2, 5 μM of the lipid peptide 24, 0.1 U / mL of the microorganism-derived transglutaminase, and 2.0 × 10 ⁵ floating cells SNU-1. Were mixed in 10 mL of D-PBS and reacted at 37 ° C. for 60 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 0.5.

[Mounting ability analysis to cell membrane (flow cytometry analysis)]
Using the lipidated protein reaction solution (including unreacted lipid peptide) obtained in Example 67, the ability of the lipidated protein to anchor to the cell membrane was evaluated. The reaction solution was washed twice with 200 μL of PBS and then suspended in 400 μL of PBS. Using the obtained suspension as a target, fluorescence per cell was quantified with a cell analyzer (product name “Cell Analyzer EC800”). The results are shown in FIG.

Example 68
[Reaction between fusion protein and lipid peptide]
The reaction between the fusion protein and the lipid peptide was carried out in the presence of the cells to be anchored with the lipidated protein.

10 μM of the fusion protein having the amino acid sequence represented by SEQ ID NO: 2, 5 μM of the lipid peptide 24, 0.1 U / mL of microorganism-derived transglutaminase, and 5.0 × 10 ⁵ floating cell SNU-1 Were mixed in D-PBS to prepare a total volume of 50 μL / well, and reacted at 37 ° C. for 30 minutes. At this time, the molar ratio between the primary amino group (here, lysine (K) residue) contained in the second peptide in the lipid peptide and the glutamine (Q) residue contained in the first peptide in the fusion protein. (K / Q ratio) was set to 0.5.

[Cytotoxicity test]
Using the lipidated protein reaction solution obtained in Example 68 (including a compound having unreacted fat-soluble vitamins), the cytotoxicity of the lipidated protein was evaluated by the WST assay. The reaction solution was washed twice with 100 μL of D-PBS, and then a color former (WST-8, manufactured by Dojindo Laboratories) diluted 6-fold with Opti-MEM was added to 110 μL / well. It incubated for 90 minutes on 37 degreeC conditions. Then, the absorbance at 450 nm was measured, and the cell viability was calculated. Cell viability was calculated using the same formula as shown in Example 1. The results are shown in FIG.

From the results of Example 67 and Example 68, the cell membrane of cells targeted for lipidated proteins can also be obtained by the method of reacting the fusion protein with the lipid peptide in the presence of cells targeted for anchoring the lipidated proteins. It has been confirmed that it can be moored at. The method of reacting the fusion protein with the lipid peptide in the presence of the cells to be moored with the lipidated protein is to synthesize and produce the lipidated protein in advance and then lipidate the cell membrane of the target cell. Compared to the method of tethering proteins, operations such as purification are unnecessary, which is beneficial.

Claims

Comprising the step of reacting a fusion protein with a compound having a primary amino group in the presence of a microorganism-derived transglutaminase to obtain a lipidated protein in which the fusion protein and the compound are cross-linked by an isopeptide bond,
The fusion protein has a first peptide and protein containing a glutamine residue,
The said compound has a lipid part or a fat-soluble vitamin part, The manufacturing method of lipidated protein.
The compound includes a second peptide containing a lysine residue and a lipid peptide having a lipid part or a fat-soluble vitamin part, wherein the primary amino group is derived from the lysine residue. 2. A method for producing a lipidated protein according to 1.
The method for producing a lipidated protein according to claim 2, wherein the second peptide has a higher ratio of hydrophilic amino acid residues than the first peptide.
The lipidation according to claim 2 or 3, wherein the second peptide further contains at least one amino acid residue selected from the group consisting of a histidine residue, a proline residue, a tryptophan residue, and an arginine residue. A method for producing a protein.
The method for producing a lipidated protein according to any one of claims 1 to 4, wherein the lipid part has an aliphatic hydrocarbon group having 12 to 18 carbon atoms.
The first peptide is at least one hydrophobic substance selected from the group consisting of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a tyrosine residue, a tryptophan residue, and a phenylalanine residue. The method for producing a lipidated protein according to any one of claims 1 to 5, further comprising a functional amino acid residue.
A fusion protein part having a first peptide and protein containing a glutamine residue, and a lipid part or a fat-soluble vitamin part;
The lipidated protein, wherein the lipid part or the fat-soluble vitamin part is bound to the fusion protein part through a glutamine residue of the first peptide and an isopeptide bond.
The lipid part or the fat-soluble vitamin part includes a lipid peptide part having a second peptide containing a lysine residue,
The lipidated protein according to claim 7, wherein the isopeptide bond is a bond between the lysine residue and the glutamine residue.
The lipidated protein according to claim 8, wherein the second peptide has a higher ratio of hydrophilic amino acid residues than the first peptide.
The lipidation according to claim 8 or 9, wherein the second peptide further contains at least one amino acid residue selected from the group consisting of a histidine residue, a proline residue, a tryptophan residue, and an arginine residue. protein.
The lipidated protein according to any one of claims 7 to 10, wherein the lipid part has an aliphatic hydrocarbon group having 12 to 18 carbon atoms.
The first peptide is at least one hydrophobic substance selected from the group consisting of a glycine residue, an alanine residue, a valine residue, a leucine residue, an isoleucine residue, a tyrosine residue, a tryptophan residue, and a phenylalanine residue. The lipidated protein according to any one of claims 7 to 11, further comprising a sex amino acid residue.