JP6090738B2 - Method for producing glycopeptide, method for producing glycoamino acid, and method for producing glycoprotein - Google Patents

Description

  The present invention relates to a method for producing a glycopeptide, a method for producing a sugar amino acid, and a method for producing a glycoprotein.

  In recent years, glycoproteins including glycoprotein preparations such as erythropoietin (EPO) and IgG have been put into practical use. Glycoproteins are mainly produced by a production method using genetic recombination (Patent Document 1), organic chemical synthesis (Non-Patent Document 1), or the like.

  In the case of the production method using gene recombination, it is known that the type of sugar chain that binds to the recombinant protein differs depending on the host in which the recombinant protein is expressed, and thus the activity on mammals differs ( Non-patent document 2). For example, when expressed in E. coli, the EPO has no sugar chain and therefore has no activity on mammals and is degraded in blood. In addition, when expressed in yeast or insect cells, it has a sugar chain different from that of the mammalian type, so that it does not have the aforementioned activity and is degraded in the liver or the like. In contrast, when expressed in mammalian cells, the EPO has a mammalian sugar chain to which sialic acid is bound, and has the aforementioned activity. However, the production method using the mammal-derived cells has a problem that the cost is high and sugar chains are not uniform.

  On the other hand, in the case of a production method utilizing organic chemical synthesis, at present, sugar chains that can be produced are limited to double-chain sugar chains. In the pheasant bird egg, there is a double-chain sugar chain to which sialic acid is bonded, but there is no sugar chain having three or more branched chains in which sialic acid is bonded to each branched chain. (Non-Patent Documents 3 and 4). For this reason, there is a demand for a means for supplying a large amount of glycoproteins bound with three- or four-strand sugar chains bound with sialic acid.

Japanese Examined Patent Publication No. 7-68272

Hirano et al., Angew. Chem. Int. Ed. 2009, Vol. 48, p. 9557-9560 Takeuchi et al., Proc. Natl. Acad. Sci. USA, 1989, Vol. 86, p. 7819-7822 Sumiyoshi et al., Biosci. Biotechnol. Biochem. 2009, Vol. 73, no. 3, p. 543-551 Sumiyoshi et al., Biosci. Biotechnol. Biochem. 2010, Vol. 74, no. 3, p. 606-613

  An object of this invention is to provide the manufacturing method of the glycopeptide which can supply the glycopeptide which has a sugar chain required for manufacture of glycoprotein cheaply and in large quantities. Another object of the present invention is to provide a method for producing a sugar amino acid capable of supplying a large amount of a sugar amino acid having a sugar chain necessary for producing a glycoprotein at a low cost. Furthermore, an object of this invention is to provide the manufacturing method of glycoprotein which can supply glycoprotein cheaply and in large quantities.

The method for producing a glycopeptide of the present invention has a glycopeptide separation step of separating the glycopeptide from an egg of an Emu ( Dromaius novae Hollandiae ), wherein the sugar of the glycopeptide is a sugar chain to which sialic acid is bound. Features.

  The method for producing a sugar amino acid according to the present invention includes a sugar amino acid separation step for separating the sugar amino acid from the glycopeptide, wherein the glycopeptide is obtained by the method for producing a glycopeptide according to the present invention. It is characterized by being a glycopeptide.

  The method for producing a glycoprotein of the present invention includes a sugar chain binding step for binding a sugar chain to a protein. As the sugar chain, the sugar chain of the glycopeptide produced by the method of producing a glycopeptide of the present invention and the present invention. It is characterized by using at least one sugar chain of the sugar amino acid sugar chain produced by the method for producing sugar amino acids.

  According to the method for producing a glycopeptide of the present invention, a glycopeptide having a sugar chain necessary for producing a glycoprotein can be supplied at a low price and in large quantities. Moreover, according to the method for producing a sugar amino acid of the present invention, a sugar amino acid having a sugar chain necessary for producing a glycoprotein can be supplied at a low cost and in a large amount. Furthermore, according to the glycoprotein production method of the present invention, since at least one of the glycopeptide produced by the glycopeptide production method of the present invention and the glycoamino acid produced by the glycoamino acid production method of the present invention is used, It is possible to supply glycoproteins at low cost and in large quantities.

FIG. 1 is a reverse phase HPLC profile of the glycopeptide fraction in Example 1. FIG. 2 is an anion exchange HPLC profile of the sugar chain fraction in Example 1. FIG. 3 is a size fraction HPLC profile of the sugar chain fraction in Example 1. 4 is a reverse phase HPLC profile of the sugar chain fraction in Example 1. FIG. FIG. 5 is a reverse-phase HPLC profile of a sugar chain digested with sialidase in Example 1. 6 is a reverse phase HPLC profile of the sugar chain fraction in Example 2. FIG. FIG. 7 is a size fraction HPLC profile of the sugar chain fraction in Example 2. FIG. 8 is a mass spectrometry profile of the sugar amino acid fraction in Example 2.

  In the glycopeptide production method of the present invention, the glycopeptide separation step is preferably a step of separating the glycopeptide from the egg white water-soluble fraction of the egg.

  In the method for producing a glycopeptide of the present invention, the sugar chain of the glycopeptide has 3 or 4 branched chains, and sialic acid is bonded to all the 3 or 4 branched chains. preferable.

In the method for producing a glycopeptide of the present invention, it is preferable that the sugar chain of the glycopeptide is represented by the following chemical formula (1) or (2).

  In the method for producing a sugar amino acid of the present invention, the sugar amino acid is preferably a sugar amino acid in which asparagine is bound to a sugar chain.

  In the glycoprotein production method of the present invention, the glycoprotein may be a glycoprotein preparation. In this case, the glycoprotein preparation is preferably erythropoietin (EPO), blood coagulation factor, growth factor, IgG, interleukin, or interferon.

<Method for producing glycopeptide>
As described above, the method for producing a glycopeptide of the present invention is a glycopeptide separation in which the sugar of the glycopeptide is a sugar chain to which sialic acid is bound, and the glycopeptide is separated from an egg of Emu ( Dromaius novaehollandiae ). It has the process. The glycopeptide production method of the present invention is characterized in that the glycopeptide is separated from the eggs of the emu in the glycopeptide separation step, and other steps are not limited at all.

  Examples of eggs of the emu include whole egg, egg yolk, egg white, defatted egg yolk, powdered egg yolk, powdered egg white, and the like, preferably egg white. As the eggs of the emu, for example, commercially available products may be used, or eggs obtained by breeding the emu and laying eggs may be used.

  The type of sugar chain of the glycopeptide is not particularly limited, and examples thereof include monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and oligosaccharides and polysaccharides are preferable. In the case of the oligosaccharide or polysaccharide, examples of the sugar chain of the glycopeptide include an N-linked sugar chain, an O-linked sugar chain, and the like, and preferably an N-linked sugar chain.

  The sugar residue constituting the sugar chain of the glycopeptide is not particularly limited. For example, glucose, galactose, mannose, glucosamine, N-acetylglucosamine, galactosamine, N-acetylgalactosamine, fucose, xylose, N-acetylneuramin An acid, N-glycolylneuraminic acid, etc. are mentioned. The sugar residue may be modified, for example. The modification is not particularly limited, and examples thereof include modification by esterification, acylation, amination, etherification, nitration, hydrolysis, dehydration reaction, oxidation-reduction, and the like.

  As described above, sialic acid is bound to the sugar chain of the glycopeptide. Sialic acid is a substance in which the functional group of neuraminic acid ((4S, 5R, 6R, 7S, 8R) -5-amino-4,6,7,8,9-pentahydroxy-2-oxononanoic acid) is substituted. Specifically, for example, the aforementioned N-acetylneuraminic acid, N-glycolylneuraminic acid and the like can be mentioned. The sugar chain to which the sialic acid is bonded is generally called a mammalian sugar chain. In the sugar chain of the glycopeptide, the number of sialic acids is not particularly limited, and may be one, for example, or two or more. The binding site of sialic acid in the sugar chain of the glycopeptide is not particularly limited. For example, the non-reducing end of the sugar chain of the glycopeptide is preferable.

  The shape of the sugar chain of the glycopeptide may be, for example, a linear type or a branched type, but a branched type is preferred. When the sugar chain of the glycopeptide is a branched chain type, the number of the branched chains is, for example, 2 to 5, preferably 3 to 4, and more preferably 4. In the present invention, the branched chain refers to, for example, a sugar chain branched from the main chain. When the sugar chain of the glycopeptide is a branched chain type, the sialic acid may be bonded to all branched chains or may be bonded to some branched chains. The sugar chain of the glycopeptide is, for example, a 4-chain sugar chain in which 1 to 4 sialic acids are bonded, a 3-chain sugar chain in which 1 to 3 sialic acids are bonded, or 2 in which 1 or 2 sialic acids are bonded. An example of this is a sugar chain. In the sugar chain of the glycopeptide, for example, the sialic acid is preferably bonded to all the branched chains, and more preferably bonded to the non-reducing ends of all the branched chains. The sugar chain of the glycopeptide preferably has 3 or 4 branched chains, and sialic acid is preferably bonded to all the 3 or 4 branched chains. By binding the sialic acid, degradation (metabolism) of the sugar chain of the glycopeptide is suppressed in vivo. In addition, the greater the number of sialic acids bound, the more the degradation in vivo of the glycoprotein to which the glycopeptide sugar chain is bound is suppressed.

Specific examples of the sugar chain to which the sialic acid is bonded include a sugar chain represented by the following chemical formula (1) or (2).

  The molecular formula of the sugar chain of the chemical formula (1) is Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-4) Manα1-3 (Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-6) 6) It is represented by Manβ1-4GlcNAcβ1-4GlcNAc. The sugar chain of the chemical formula (1) has four uniform branched chains, and sialic acid is bonded to the non-reducing end of each branched chain. The molecular formula of the sugar chain of the chemical formula (2) is Siaα2-3Galβ1-4GlcNAcβ1-2 (Siaα2-3Galβ1-4GlcNAcβ1-4) Manα1-3 (Siaα2-3Galβ1-4GlcNAcβ1-2Manα1-6) Manβ1-4GlcNAcβ1-4GlcNAcβ1-4 Represented. The sugar chain of the chemical formula (2) has three uniform branched chains, and sialic acid is bonded to the non-reducing end of each branched chain.

  Next, an example of the manufacturing method of the glycopeptide of this invention is demonstrated.

  First, the emu egg is prepared and divided into egg white and egg yolk. On the other hand, a mixed solvent of methanol and chloroform is prepared as a solvent. The egg white and an equal volume of the mixed solvent are mixed and homogenized. This mixed solution is centrifuged, and the aqueous phase is recovered from the aqueous phase, the organic phase, and the interface layer formed between the aqueous phase and the organic phase. The aqueous phase is lyophilized, denatured with urea, and reductively alkylated with 2-mercaptoethanol and 4-vinylpyridine lysyl. The modification and reductive alkylation using urea are not essential steps and may not be performed. After excess reagent is removed by dialysis, enzymatic degradation is performed to obtain a glycopeptide fraction. Examples of the enzyme include endopeptidase, pronase, trypsin, hepsin, thermolysin and the like. The decomposition conditions can be appropriately set according to conditions such as the type of enzyme and the amount of treatment. The glycopeptide fraction may contain components other than the glycopeptide, for example. The component is not particularly limited.

  The solvent is not limited to the above-mentioned mixed solvent of methanol and chloroform, and may be any solvent that can fractionate the glycopeptide fraction. Examples of the solvent include an aqueous solvent and an organic solvent. Examples of the aqueous solvent include water. Examples of the organic solvent include methanol, chloroform, acetone, dichloromethane, dichloroethane, and the like. The solvent may be one type or a mixed solvent in which two or more types are combined. Examples of the mixed solvent include a mixed solvent of water-saturated phenol and chloroform other than the above-described mixed solvent of methanol and chloroform. In the mixed solvent, the volume ratio of each solvent can be appropriately set according to the polarity of the glycopeptide and is not particularly limited.

  The glycopeptide thus obtained is, for example, a peptide in which a sugar chain is bound to asparagine of a peptide composed of several tens of amino acids.

<Method for producing sugar amino acid>
Next, the method for producing a sugar amino acid of the present invention can be carried out, for example, by enzymatically decomposing a glycopeptide obtained as described above and separating the sugar amino acid.

  Examples of the enzyme include protease and peptidase. The protease is not particularly limited, and examples thereof include exoprotease and endoprotease. Specifically, pronase (trade name), lysyl endopeptidase (registered trademark), thermolysin (trade name), V8 protease (trade name) ), Protease M “Amano” (trade name), Samoaze (trade name) and the like. The peptidase is not particularly limited, and examples thereof include carboxypeptidase, aminopeptidase, and endopeptidase. Specifically, carboxypeptidase Y (trade name), aminopeptidase I (trade name), peptidase R (trade name) ) And the like. One type of enzyme may be used at the time of the enzymatic decomposition, or two or more types may be combined. Moreover, when using a some enzyme at the time of the said enzyme decomposition | disassembly, you may decompose | disassemble with a some enzyme simultaneously, and may decompose | disassemble sequentially with one enzyme at a time. The degradation conditions can be appropriately set according to conditions such as the type of the enzyme and the amount of the glycopeptide. The sugar amino acid fraction may contain components other than sugar amino acids, for example. The component is not particularly limited.

  The sugar amino acid thus obtained is, for example, one in which a sugar chain is bound to asparagine.

<Method for producing glycoprotein>
Next, the method for producing a glycoprotein of the present invention can be carried out, for example, by adding at least one sugar chain of the glycopeptide and the glycoamino acid obtained as described above to the protein.

  The glycoprotein includes at least one sugar chain of the glycopeptide produced by the glycopeptide production method of the present invention and the sugar amino acid sugar chain produced by the glycoamino acid production method of the present invention. For this reason, the glycoprotein has, for example, high in vivo stability and high activity against the living body.

  Next, an example of the method for producing the glycoprotein of the present invention will be described.

  First, the glycopeptide or the sugar amino acid is prepared. The glycopeptide and the sugar amino acid can be prepared, for example, as described above.

  Next, the protein is prepared. Examples of the protein include a recombinant protein. Specifically, the recombinant protein is prepared by introducing a DNA fragment encoding a desired protein into a host to produce a transformant, culturing the transformant, collecting the culture, It can be obtained by purification.

  The protein is not particularly limited, and examples thereof include erythropoietin (EPO), blood coagulation factor, growth factor, IgG, interleukin, and proteins constituting interferon. The interferon is not particularly limited, and examples thereof include IFN-α, IFN-β, IFN-ω, IFN-ε, IFN-κ, IFN-γ, and IFN-λ. Although it does not restrict | limit especially as said interleukin, For example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18 and the like.

  The host is not particularly limited, and examples thereof include Escherichia coli, yeast, filamentous fungi, actinomycetes, COS cells, CHO cells, and Sf9 cells. The E. coli is not particularly limited, and examples thereof include strains such as S17-1, BL21 (DE3), BL21-CodonPlus (DE3) -RIL, Rosetta2 (DE3), Rosetta-gami2 (DE3).

  In the protein production process, methods for introducing a DNA fragment into the host, culturing the transformant, recovering and purifying the culture are not particularly limited, and for example, conventionally known methods can be appropriately employed. In the present invention, the method for producing the protein is not limited to the above-described method for producing a recombinant protein, and conventionally known methods such as a solid phase synthesis method can be appropriately employed.

  A glycoprotein is obtained by linking at least one sugar chain of a sugar chain of the glycopeptide and a sugar amino acid with the protein using native chemical ligation. When native chemical ligation is used, cysteine is preferably added when the N-terminus of the glycopeptide and the sugar amino acid is other than cysteine. In native chemical ligation, when a thioester is added to the N-terminal cysteine of at least one of the glycopeptide and glycoamino acid, the sulfhydryl group of cysteine first undergoes a nucleophilic attack on the carboxy group, causing transesterification, and then adjacent. This is a reaction in which an amino group nucleophilically attacks a carbonyl group and a sulfhydryl group is eliminated to form a peptide bond.

  The glycoprotein thus obtained can be used, for example, as a drug or a physiologically active substance. Examples of the glycoprotein preparation include the aforementioned erythropoietin (EPO), blood coagulation factor, growth factor, IgG, interleukin, interferon and the like to which a sugar chain is bound. The interferon is not particularly limited, and examples thereof include the aforementioned interferon. It does not restrict | limit especially as said interleukin, For example, the above-mentioned interleukin is mentioned.

  The glycoprotein can also be used for applications other than glycoprotein preparations such as sugar chain antigens and sugar chain probes. Examples of the sugar chain antigen and sugar chain probe include substances in which a large amount of sugar chains are bound to the protein. These can be used, for example, to deliver a drug specifically to an organ or tissue (missile therapy).

  In the glycoprotein, the sugar chain includes, in addition to the glycopeptide produced by the glycopeptide production method of the present invention and the sugar amino acid sugar chain produced by the glycoamino acid production method of the present invention, A sugar chain may be used.

  The type, sugar residue, modification, shape, number of sugar chains and the like of the other sugar chains are not particularly limited. For example, in the method for producing a glycopeptide or the sugar amino acid in the method for producing a glycopeptide, It is the same as the sugar chain of a sugar amino acid. For example, sialic acid may or may not be bound to the other sugar chain, but the former is preferably the former. The method for producing the other sugar chain is not particularly limited, and for example, a conventionally known method can be appropriately employed.

  In the sugar chain binding step, only one of the glycopeptide and the sugar amino acid may be bound to the protein or both may be bound, but in order to reduce antigenicity in vivo, It is preferred to bind only sugar amino acids.

  In the present invention, the method for binding a sugar chain to the protein is not limited to the above-described method. For example, only a sugar chain is separated from the glycopeptide or sugar amino acid, and only the sugar chain is bound to the protein. Conventionally known methods can be appropriately employed.

  Next, examples of the present invention will be described. However, the present invention is not limited by the following examples.

Emu ( Dromaius novaehollandia ) eggs (manufactured by Changnan Green System) were separated into egg yolk and egg white. An extract obtained by mixing methanol and chloroform at 1: 1 (volume ratio) with the egg white was mixed in an equal volume and homogenized. The mixture was centrifuged at 3000 × g (g is gravitational acceleration) for 20 minutes, and the aqueous phase was recovered. This aqueous phase was freeze-dried, 20 mg of which was denatured with 6 mol / L urea and then reductively alkylated with 2-mercaptoethanol and 4-vinylpyridine lysyl. After excess reagent was removed by dialysis, enzymatic digestion was performed using lysyl endopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.) to obtain a glycopeptide.

  The glycopeptide was subjected to gel filtration under the following conditions, and the portion colored with anthrone sulfate was collected to obtain a glycopeptide fraction.

(Purification conditions for gel filtration)
(1) Detection equipment: Spectrophotometer (manufactured by Shimadzu Corporation)
(2) Column: Glass tube (inner diameter 2.6 mm × 40 cm)
(3) Gel filtration resin: Biogel P-4 (manufactured by Bio-Rad Laboratories)
(4) Column equilibration buffer: water (5) Mobile phase: solvent water
Flow rate 50mL / h
(6) Preparative volume: 3.8 mL / fraction

  The glycopeptide fraction was subjected to reverse phase HPLC under the following conditions.

(Reverse phase HPLC analysis conditions)
(1) Analytical instrument: HPLC system with an absorbance meter (manufactured by Shimadzu Corporation)
(2) Column: Product name: Cosmosil 5C18-P
(Inner diameter 4 mm x 25 cm, manufactured by Nacalai Tesque)
(3) Column equilibration buffer A: 0.01% TFA aqueous solution
B: Acetonitrile
+ 0.01% TFA aqueous solution (4) Elution condition: Time (solvent ratio (A: B))
0 min: (A: B = 100: 0)
60 minutes: (A: B = 0: 100)
The ratio of solvent A linearly from 0 to 60 minutes so that
Elution, increasing the proportion of solvent B and elution.
Flow rate 1mL / min

  FIG. 1 shows the reverse phase HPLC profile of the glycopeptide fraction. In FIG. 1, the vertical axis represents absorbance at a wavelength of 210 nm, and the horizontal axis represents retention time. A fraction obtained by fractionating the glycopeptides of the peaks (1, 2 and 3) indicated by the arrows in FIG. 1 and a fraction obtained by fractionating the glycopeptides of peaks 2 and 3 were prepared.

The three peaks of glycopeptides separated by reversed-phase HLPC were found to be four peptides having the following amino acid sequences by peptide analysis. It was also revealed that a sugar chain was bound to asparagine (N). The peptide (4) was obtained when the fractions of peaks 2 and 3 were collected.
(1) CDFCNVVESNGTRTLGHFG (SEQ ID NO: 1)
(2) YPNTTNEDGK (SEQ ID NO: 2)
(3) YANATNEEEGK (SEQ ID NO: 3)
(4) ILNPICGSDGVTYSNDLLCAYNIEYGANVSK (SEQ ID NO: 4)

  As a result of homology search of these peptides, it was found that they were glycopeptides possessed by the glycoprotein present in the Emu egg.

  The three peaks of glycopeptides separated by reverse phase HPLC were each subjected to hydrazine decomposition, N-acetylation, and pyridylamination, whereby the sugar chain was excised from the glycopeptide and fluorescently labeled. After removing the excess reagent, it was subjected to anion exchange HPLC under the following conditions to separate by the number of sialic acids, and fractions from which tetrasialylated sugar chains were eluted were collected.

(Analysis conditions for anion exchange HPLC)
(1) Analytical instrument: HPLC system with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: Product name Mono Q
(Manufactured by GE Healthcare)
(3) Buffer for buffer equilibration: ammonia water (pH 9.0)
(4) Mobile phase: solvent A: aqueous ammonia
B: 0.5 mol / L acetic acid-ammonia buffer (pH 9.0)
Gradient condition: time (solvent ratio (A: B))
0 minutes (A: B = 100: 0)
10 minutes (A: B = 100: 0)
40 minutes (A: B = 60: 40)
Flow rate 1mL / min

  The tetrasialylated sugar chain fraction was again subjected to the aforementioned anion exchange HPLC. FIG. 2 shows the anion exchange HPLC profile of the tetrasialylated sugar chain fraction. In FIG. 2, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time. Each peak indicated by 4SiaTetra in FIG. 2 (a fraction from which tetrasialylated sugar chains are eluted) was collected.

  The tetrasialylated sugar chain fraction separated by the anion exchange HPLC was subjected to size fractionation HPLC under the following conditions.

(Analysis conditions for size fractionation HPLC)
(1) Analytical instrument: HPLC with fluorescence detector (manufactured by Shimadzu Corporation)
(2) Column: Trade name TSK gel Amido80
(Inner diameter 2.0 mm x 25 cm, manufactured by Tosoh Corporation)
(3) Buffer solution for column equilibration: mixed solvent of A and B (solvent ratio (A: B) = 8: 2)
A: Acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
(4) Mobile phase: solvent A: acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
Gradient condition: time (solvent ratio (A: B))
0 min: (A: B = 80: 20)
10 minutes: (A: B = 70: 30)
35 minutes: (A: B = 30: 70)
Flow rate 0.18mL / min

  FIG. 3 shows the size fraction HPLC profile of the tetrasialylated sugar chain fraction. In FIG. 3, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time. Each peak (elution fraction of tetrasialylated sugar chain) indicated by 4SiaTetra in FIG. 3 was collected.

  The tetrasialylated sugar chain fraction separated by the size fractionation HPLC was subjected to the aforementioned reverse phase HPLC. FIG. 4 shows the reverse phase HPLC profile of the tetrasialylated sugar chain fraction. In FIG. 4, the vertical axis represents fluorescence intensity, and the horizontal axis represents retention time. Each peak (elution fraction of tetrasialylated sugar chain) indicated by 4SiaTetra in FIG. 4 was collected.

  The sugar chain obtained by digesting the tetrasialylated sugar chain fraction separated by the reverse phase HPLC with α2,3-sialitase was subjected to the above-described reverse phase HPLC. FIG. 5 shows the reverse phase HPLC profile of the sugar chain. In FIG. 5, the vertical axis represents the fluorescence intensity, and the horizontal axis represents the retention time. As shown in FIG. 5, sialic acid was removed and each peak indicated by Tetra was detected. From this, it was found that in the sugar chain, four sialic acids were bonded to tetra at α2,3.

[Example 2]
The sugar amino acid was separated from the glycopeptide fractionated in Example 1 and purified.

The following proteases were dissolved in 50 mmol / L Tris-HCl buffer (pH 8.0) so as to be 0.1 mg / mL to prepare six types of protease solutions.
Pronase (trade name, Boehringer Mannheim, Merck, Calbiochem)
Lysyl endopeptidase (registered trademark, manufactured by Wako Pure Chemical Industries, Ltd.)
Thermolysin (trade name, manufactured by Nacalai Tesque)
V8 protease (trade name, manufactured by Roche)
Protease M “Amano” (trade name, manufactured by Amano Enzam)
Samoaise (trade name, manufactured by Amano Enzam)

  50 mg of the glycopeptide obtained in Example 1 and 10 mL of the protease solution (equivalent to 1 mg of protease) were mixed and reacted. The reaction between the glycopeptide and the protease solution was sequentially performed one by one. The reaction conditions of the glycopeptide and the protease solution were all reacted at 37 ° C. for 17 hours.

  After the reaction, it was subjected to gel filtration under the following conditions, and the portion colored with methanolic sulfuric acid was collected to obtain a fraction containing sugar.

(Purification conditions for gel filtration)
(1) Detector: Spectrophotometer (Amersham)
(2) Column: Glass tube (3) Gel filtration resin:
Biogel P-4 (manufactured by Bio-Rad Laboratories)
(4) Column equilibration buffer:
Water (5) Mobile phase: Solvent Water
Flow rate 10mL / h
(6) Preparative volume: 1 mL / fraction

Next, the following peptidases were dissolved in a 50 mmol / L phosphate buffer (pH 6.0) so as to be 0.1 mg / mL to prepare three types of peptidases.
Carboxypeptidase Y (trade name, manufactured by Oriental Yeast Co., Ltd.)
Aminopeptidase I (trade name, manufactured by Takara Bio Inc.)
Peptidase R (trade name, manufactured by Amano Enzam)

  The fraction containing the sugar and 10 mL of the peptidase solution (equivalent to 1 mg of peptidase) were mixed and reacted. The reaction between the glycopeptide and the peptidase solution was sequentially performed one by one. The reaction conditions of the glycopeptide and the peptidase solution were all reacted at 37 ° C. for 17 hours.

  After the reaction, it was subjected to gel filtration again under the gel filtration purification conditions of Example 2, and a portion colored with methanolic sulfuric acid was collected to obtain a fraction containing sugar.

  The fraction containing sugar was centrifuged at 30 ° C. and 2,000 rpm for 8 hours and concentrated. After the concentration, a fraction colored with methanol sulfate and ninhydrin reagent was collected to obtain a sugar amino acid fraction.

  The sugar amino acid was hydrolyzed with hydrazine, N-acetylated, and then pyridylaminated to cut out the sugar chain from the sugar amino acid and fluorescently label the sugar chain. After the fluorescent labeling, excess reagent was removed.

  The fluorescently labeled sugar chain was subjected to reverse phase HPLC under the following conditions.

(Reverse phase HPLC analysis conditions)
(1) Analytical instrument: 2475 multi-wavelength fluorescence detector (manufactured by Waters)
321 pump (Giloson)
(2) Column: Product name: Cosmosil 5C18-P
(Inner diameter 4.6 mm x 150 mm, manufactured by Nacalai Tesque)
(3) Column equilibration buffer: A and B mixed solvent (solvent ratio (A: B) = 95: 5)
A: 50 mmol / L acetic acid-ammonia buffer (pH 4.0)
B: 50 mmol / L acetic acid-ammonia buffer (pH 4.0)
+ 0.5% 1-butanol (4) Mobile phase: Solvent A: 50 mmol / L acetic acid-ammonia buffer (pH 4.0)
B: 50 mmol / L acetic acid-ammonia buffer (pH 4.0)
+ 0.5% 1-butanol
Gradient condition: time (solvent ratio (A: B))
0 min: (A: B = 95: 5)
55 minutes: (A: B = 0: 100)
The proportion of solvent A linearly from 0 to 55 minutes
The ratio of solvent B was increased.
Flow rate 1.5mL / min
(5) Excitation wavelength: 320 nm
Fluorescence wavelength: 400nm
(6) Column temperature: 25 ° C

  FIG. 6 shows the reverse phase HPLC profile of the fluorescently labeled sugar chain. In FIG. 6, the vertical axis represents fluorescence intensity at 420 nm, the horizontal axis represents retention time, the arrow in FIG. 6 and 4SiaTetra-PA represent pyridylaminated tetrasialyl sugar chains (PA-modified tetrasialyl sugar chains). As shown in FIG. 6, since a peak indicating a PA-modified tetrasialyl sugar chain was detected, it was found that the sugar chain of the sugar amino acid was a tetrasialyl sugar chain.

  The fluorescently labeled sugar chain was subjected to size fractionation HPLC under the following conditions.

(Analysis conditions for size fractionation HPLC)
(1) Analytical instrument: 2475 multi-wavelength fluorescence detector (manufactured by Waters)
321 pump (Giloson)
(2) Column: Product name TSK gel Amido80
(Inner diameter 4.6 mm x 100 mm, manufactured by Tosoh Corporation)
(3) Buffer solution for column equilibration: mixed solvent of A and B (solvent ratio (A: B) = 8: 2)
A: Acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
(4) Mobile phase: solvent A: acetonitrile
B: 50 mmol / L formic acid-ammonia buffer (pH 4.4)
Gradient condition: time (solvent ratio (A: B))
0 min: (A: B = 80: 20)
3 minutes: (A: B = 70: 30)
35 minutes: (A: B = 35: 65)
The proportion of solvent A linearly from 0 to 35 minutes
The ratio of solvent B was increased.
Flow rate 0.6mL / min
(5) Excitation wavelength: 320 nm
Fluorescence wavelength: 400nm
(6) Column temperature: 25 ° C

  FIG. 7 shows the size fraction HPLC profile of the fluorescently labeled sugar chain. In FIG. 7, the vertical axis represents the fluorescence intensity at 420 nm, the horizontal axis represents the retention time, the arrow in FIG. 7 and 4SiaTetra-PA represent the PA-modified tetrasialyl sugar chain. As shown in FIG. 7, since a peak indicating a PA-modified tetrasialyl sugar chain was detected, it was found that the sugar chain of the sugar amino acid was a tetrasialyl sugar chain.

(Mass spectrometry)
1 μL of the sugar amino acid fraction and 1 μL of an ethanol solution were mixed, and the mixed solution was placed on AnchorChip (manufactured by Bruker) and dried. As the ethanol solution, a solution in which 2,5-dihydroxybenzoic acid was dissolved in 30% ethanol to 1 mg / mL was used. After the drying, mass analysis of the sugar amino acid fraction on the chip was performed using MALDI-TOF MS ultraflexstream ^™ (manufactured by Bruker).

  FIG. 8 shows the mass spectrometry profile of the sugar amino acid fraction. In FIG. 8, the vertical axis represents peak intensity (Response), the horizontal axis represents molecular weight / charge number (M / z), and the arrows and Tetra-Asn + Na in FIG. The number inside is its molecular weight. As shown in FIG. 8, since a peak indicating tetrasaccharide chained asparagine was detected, it was found that the sugar amino acid was tetrasaccharide chained asparagine. Further, as shown by the results of the reverse phase HPLC in FIG. 6 and the size fractionation HPLC in FIG. 7, since the sialic acid is bound to the tetrasaccharide chain, the sugar amino acid is tetrasialylglycosylated asparagine. I found out that

  According to the present invention, glycopeptides having a sugar chain necessary for the production of glycoprotein can be supplied at low cost and in large quantities. In addition, according to the present invention, sugar amino acids having sugar chains necessary for the production of glycoprotein can be supplied at low cost and in large quantities. Therefore, according to the present invention, glycoproteins can be supplied at low cost and in large quantities. Therefore, the present invention is applicable to a wide range of fields such as the medical field, and the field of application is not limited.

Claims (5)

A method for producing a glycopeptide comprising:
The sugar of the glycopeptide is a sugar chain to which sialic acid is bound,
A glycopeptide separation step of separating the glycopeptide from an egg of an emu ( Dromaius novaehollandiae ) ,
The sugar chain is represented by the following chemical formula (1) or (2):
The glycopeptide separation step is a step of mixing the egg white of the emu egg and a solvent containing an organic solvent;
Separating the resulting mixture into an aqueous phase, an organic phase and an interface layer formed between the aqueous and organic phases; and
A production method comprising the step of separating the glycopeptide by recovering the aqueous phase .
A method for producing a sugar amino acid in which an amino acid is bonded to a sugar chain ,
Includes sugar acids separation step of separating the sugar amino acid is asparagine glycopeptide sugar chain bound,
The production method according to claim 1, wherein the glycopeptide is a glycopeptide obtained by the production method according to claim 1 . A method for producing a glycoprotein comprising:
Including a sugar chain binding step of binding a sugar chain to a protein,
As the sugar chain, and characterized by using at least one of sugar chains of sugar chains of oligosaccharides and claim 2 sugar acids produced by the method according glycopeptides produced by the method of claim 1, wherein Manufacturing method. The production method according to claim 3 , wherein the glycoprotein is a glycoprotein preparation. The production method according to claim 4 , wherein the glycoprotein preparation is erythropoietin (EPO), blood coagulation factor, growth factor, IgG, interleukin or interferon.