JPWO2015029911A1 - Recombinant protein, recombinant gene, recombinant vector, transformant, method for producing pyruvate-containing complex type sugar chain, method for producing pyruvate-containing complex type sugar chain-modified protein, pyruvate-containing complex type sugar chain and pyruvate-containing complex type Glycoprotein - Google Patents

Abstract

The present invention relates to a sugar chain that has a complex sugar chain structure and is not easily affected by blood degradation enzymes, a glycoprotein having the sugar chain, a method for producing the sugar chain and the glycoprotein, and Recombinant proteins, recombinant genes, recombinant vectors and transformants used are provided. The recombinant protein of the present invention is the following (a) or (b). (A) a protein consisting of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; A protein comprising an amino acid sequence in which several amino acids have been deleted, inserted, substituted or added, and having galactose-specific pyruvate transfer activity.

Description

  The present invention relates to a recombinant protein, a recombinant gene, a recombinant vector, a transformant, a method for producing a pyruvate-containing complex sugar chain, a method for producing a pyruvate-containing complex sugar chain-modified protein, a pyruvate-containing complex sugar chain and pyruvate The present invention relates to a complex sugar chain-modified protein.

  Complex type sugar chains are added to all proteins in blood produced by higher animals. Sialic acid is added to the complex sugar chain, and sialic acid is removed by weak sialidase activity in blood. A lectin that recognizes galactose in the liver binds to the exposed galactose by removing sialic acid, and determines the life span of glycoprotein in blood.

  Therefore, glycoproteins such as hormones introduced into the blood must be administered every few weeks. For erythropoietin, which is actually a glycoprotein hormone, many attempts have been made to increase the residence time in the blood by increasing the number of added sugar chains or by treating the sugar chains with polyethylene glycol. (See Non-Patent Document 1). Thus, there is a demand for sugar chains that can be applied to glycoprotein pharmaceuticals and have a long residence time in blood.

  Fission yeast, which is a eukaryotic microorganism, has the ability to add sugar chains to proteins in the same way as humans. The sugar chain of fission yeast is almost the same as the human sugar chain and its constituent components, but it is not useful as a protein drug because it has a sugar chain structure different from that of the human sugar chain and may have antigenicity. There is a problem.

Eliot S, Pham E, Macudogal IC: Erythropoietins: a common mechanism of action. Exp Hematol 36, 1573-1584 (2008)

  The present invention has been made in view of the above circumstances, and has a structure of a complex type sugar chain and is hardly affected by a degrading enzyme in blood, a glycoprotein having the sugar chain, the sugar chain, and the sugar chain. It is an object of the present invention to provide a method for producing a glycoprotein and a recombinant protein, a recombinant gene, a recombinant vector and a transformant used in the production method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have an amino acid sequence obtained by modifying a specific amino acid in the amino acid sequence of pyruvate transferase of the fission yeast Schizosaccharomyces pombe. By using a recombinant protein or a transformant in which the recombinant protein is expressed, pyruvate can be transferred instead of sialic acid added to galactose located at the end of the human complex sugar chain. The headline and the present invention were completed.

  That is, the present invention provides a recombinant protein, a recombinant gene, a recombinant vector, a transformant, a method for producing a pyruvate-containing complex sugar chain, a method for producing a pyruvate-containing complex sugar chain-modified protein, and pyruvate having the following characteristics: And a pyruvate-containing complex sugar chain-modified protein.

(1) The following (a) or (b), a recombinant protein:
(A) a protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13;
(B) consisting of an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added at a site other than amino acid number 168 of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; Protein with pyruvate transfer activity specific to galactose.
(2) A recombinant gene encoding any of the proteins described in (1) above.
(3) A recombinant vector comprising the gene according to (2).
(4) A transformant comprising the recombinant vector according to (3).

(5) A method for producing a pyruvic acid-containing complex type sugar chain, comprising a step of transferring pyruvic acid to galactose located at a sugar chain end using the recombinant protein according to (1).
(6) A method for producing a pyruvic acid-containing complex type sugar chain, comprising a step of transferring pyruvic acid to galactose located at the sugar chain end using the transformant according to (4).
(7) The method for producing a pyruvic acid-containing complex sugar chain according to (5) or (6) above, wherein the sugar bonded to galactose located at the sugar chain terminal is N-acetylglucosamine.
(8) The method for producing a pyruvic acid-containing complex sugar chain according to any one of (5) to (7), wherein the sugar chain terminal is a human sugar chain.

(9) A method for producing a pyruvic acid-containing complex type sugar chain-modified protein, comprising a step of transferring pyruvic acid to galactose located at the end of a sugar chain using the recombinant protein according to (1).
(10) A method for producing a pyruvic acid-containing complex type sugar chain-modified protein, comprising a step of transferring pyruvic acid to galactose located at the end of a sugar chain using the transformant according to (4).
(11) The pyruvic acid-containing complex sugar chain-modified protein according to (9) or (10), wherein the sugar bound to the protein side of galactose located at the sugar chain terminal is N-acetylglucosamine. Production method.
(12) The method for producing a pyruvic acid-containing complex type sugar chain-modified protein according to any one of (9) to (11), wherein the sugar chain terminal is a human type sugar chain.

(13) A pyruvic acid-containing complex type sugar chain comprising a sugar chain structure comprising pyruvic oxidized galactose formed by binding pyruvic acid to galactose, wherein the sugar chain structure has a basic structure of an asparagine-linked sugar chain.
(14) The pyruvic acid-containing complex sugar chain according to (13), wherein the sugar chain structure is a human sugar chain.
(15) The pyruvic acid-containing complex sugar chain according to (14), wherein the pyruvate galactose is located at a sugar chain terminal of the human sugar chain.
(16) The pyruvic acid-containing complex type sugar chain according to any one of (13) to (15), wherein the sugar bonded to the pyruvic oxidized galactose is N-acetylglucosamine.
(17) A pyruvate-containing complex sugar chain-modified protein having the pyruvate-containing complex sugar chain according to any one of (13) to (16).

  According to the present invention, a pyruvic acid-containing complex sugar chain that is very stable in blood and a glycoprotein having the sugar chain are obtained. Therefore, by applying the pyruvic acid-containing complex sugar chain of the present invention to a glycoprotein drug, it is possible to obtain a very useful glycoprotein drug whose residence time in blood has been dramatically improved.

It is a figure of the HPLC analysis result which shows the result of the pyruvate transfer in Example 1 and Comparative Example 1. 4 is a schematic diagram showing the structure of a sugar chain of PA001 used in Example 2 and Comparative Example 2. FIG. It is a figure of the HPLC analysis result which shows the result of the pyruvate transfer in the comparative example 2. It is a figure of the HPLC analysis result which shows the result of pyruvate transfer in Example 2. It is a figure of the HPLC analysis result which shows presence of pyrubin-oxidized PA001 in the reaction liquid after the reaction obtained in Example 2 by the comparison with the reaction liquid obtained by the process on a weak acid and 100 degreeC conditions. It is a figure of the HPLC analysis result which shows presence of pyrubin-oxidized PA001 in the reaction liquid after the reaction obtained in Example 2 by the comparison with the reaction liquid obtained by the beta-galactosidase treatment. It is the table | surface which put together the characteristic of the origin of the lectin fixed to the lectin array, the acquisition place, and specificity. It is the table | surface which put together the characteristic of the origin of the lectin fixed to the lectin array, the acquisition place, and specificity. It is the experimental result which added the pyrubin oxidized glycopeptide (PVGP) labeled with Cy3 in Example 3 to the lectin array. It is the experimental result which added the sialyl glycopeptide (SGP) labeled with Cy3 in Example 3 to the lectin array. It is the graph which digitized the experimental data obtained using 10 microgram / ml PVGP in Example 3. FIG. It is the graph which digitized the experimental data obtained using 1 microgram / ml PVGP in Example 3. FIG. It is the graph which digitized the experimental data obtained using 10 microgram / ml SGP in Example 3. FIG. It is the graph which digitized the experimental data obtained using 1 microgram / ml SGP in Example 3. FIG. It is a figure which shows the structural similarity of the sialic acid which couple | bonded (alpha) 2,6 with galactose, and the thing which galactose oxidized with pyrubin.

In the present invention, the “complex type sugar chain” means an N-linked sugar chain having various sugars and more N-acetylglucosamine than the original two molecules. The complex type sugar chain preferably contains galactose which is a recognition target of pyruvate transferase, and more preferably a human type sugar chain. “Human-type sugar chains” are sugar chains originally present in the human body, or one to several sugars within the range that is not antigenic to humans based on the sugar chain structure present in the human body. Alternatively, it may be a sugar chain from which a functional group is deleted or added.
The complex sugar chain preferably has “basic structure of an asparagine-linked sugar chain” in addition to galactose. The basic structure of this asparagine-linked sugar chain is represented by the following formula (1). Mannose (Man) α1 → 6 (Manα1 → 3) Manβ1 → 4N-acetylglucosamine (GlcNAc) β1 → 4GlcNAc.

The galactose in the complex type sugar chain is preferably arranged so that pyruvic acid can bind, and more preferably located at the end of the human complex type sugar chain so that pyruvic acid can bind. Moreover, it is more preferable that the galactose in the complex type sugar chain to which pyruvic acid can bind is bound to N-acetylglucosamine.
In the present specification, when the complex type sugar chain is the human type sugar chain, the sugar chain is referred to as a “human complex type sugar chain”. That is, the “human complex sugar chain” in the present invention may have a sugar chain structure represented by the above formula (1) and a sugar chain structure represented by the following formula (2).

  Specific examples of the human complex sugar chain include sugar chains represented by the following formula (3-1) to the following formula (3-3).

  In addition, the “human complex sugar chain” in the present invention may have the sugar chain structure represented by the above formula (1) and the structure of lactose (D-galactopyranoylβ1 → 4D-glucose). The lactose structure is preferably located at the end of the sugar chain structure.

<Pyruvic acid-containing complex sugar chain and glycoprotein>
In the pyruvate-containing complex sugar chain of the present invention, the complex sugar chain contains pyruvate galactose (hereinafter referred to as PvGal) formed by binding pyruvate to galactose. PvGal is a compound represented by the following formula (4).

[Wherein, R represents a hydrogen atom or a sugar. ]

  Therefore, an example of the pyruvic acid-containing complex type sugar chain of the present invention is a sugar chain represented by the following formula (5-1).

  The pyruvate-containing complex sugar chain-modified protein of the present invention is a glycoprotein having the pyruvate-containing human complex sugar chain of the present invention. A sugar chain having the “basic structure of an asparagine-linked sugar chain” represented by the above formula (1) is present bound to asparagine (Asn) constituting a protein in the human body. Therefore, the sugar chain of the pyruvate-containing complex sugar chain-modified protein of the present invention is bound to asparagine constituting the pyruvate-containing complex sugar chain-modified protein, for example, as represented by the following formula (6). Can be exemplified.

Complex type sugar chains are added to all proteins in blood produced by higher animals. Sialic acid is added to the complex sugar chain, and when this sialic acid is removed by sialidase activity, the displayed galactose becomes a so-called degradation signal of the glycoprotein, and the retention time of the glycoprotein in the blood decreases. It becomes a factor of.
Pyruvate is added to galactose contained in the pyruvate-containing complex sugar chain of the present invention instead of sialic acid. Since there is no enzyme that removes pyruvate from galactose in the blood, the residence time in the blood of the pyruvate-containing complex sugar chain-modified protein of the present invention having the pyruvate-containing complex sugar chain of the present invention has jumped. As a result, the value of glycoprotein pharmaceuticals can be greatly increased.

  The protein portion of the glycoprotein having a pyruvate-containing complex sugar chain of the present invention is not particularly limited, and may be a protein portion of a heterologous glycoprotein in addition to a protein portion of a human-derived glycoprotein. The glycoprotein of the present invention is preferably a protein that may be contained in blood in the human body from the viewpoint that the residence time in blood is improved. The function and type of glycoprotein are not particularly limited, and any enzyme, hormone, cytokine, antibody, etc. can be used. Specific examples of glycoproteins include interferon, luteinizing hormone, follicle stimulating hormone, thyroid stimulating hormone, erythropoietin, and the like.

<Recombinant protein>
The short residence time of glycoprotein in blood is partly due to the removal of sialic acid bound to galactose located at the sugar chain terminal by sialidase. Based on this, the present inventors have come up with the idea of prolonging the residence time of glycoproteins in blood by transferring another compound to galactose instead of sialic acid bound to galactose of the complex type sugar chain. It was.
Fission yeast has the ability to add sugar chains to proteins as in humans, and the types of sugars that make up the sugar chains are almost the same as the types of sugars that make up human-type sugar chains. However, pyruvic acid galactose, which does not exist in human sugar chains, exists in the sugar chains of fission yeast.
Therefore, an attempt was made to transfer pyruvic acid to galactose located at the sugar chain end of the human complex sugar chain.

A gene encoding a protein presumed to be pyruvate transferase that causes pyruvate oxidation of fission yeast galactose was named Pvg1, and was suggested by Andrew Sheva (Andreishcheva EN et al. J. Biol. Chem. 2004; 279: 35644 -35655).
However, the protein encoded by the Pvg1 gene of fission yeast has only a low homology compared to a protein presumed to be a bacterial pyruvate transferase, and it was unclear whether it actually had pyruvate transfer activity. Therefore, the present inventors purified the Pvg1 protein and measured the enzyme activity, and revealed that Pvg1 has pyruvate transferase activity (see Yoritsune K. et al. FEBS lett. (2013)).

  In the present invention, the present inventors have revealed that in the fission yeast Pvg1 protein described above, pyruvate cannot be transferred to galactose located at the end of the human complex type sugar chain (Comparative Example 2 described later). reference). Therefore, the present inventors modified Pvg1 protein, and transferred a protein consisting of the following amino acid sequences of SEQ ID NOs: 1 to 13 capable of transferring pyruvic acid to galactose located at the sugar chain terminal of the complex sugar chain. It came to choose.

The recombinant protein of the present invention is the following (a) or (b).
(A) a protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13;
(B) consisting of an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added at a site other than amino acid number 168 of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; Protein with pyruvate transfer activity specific to galactose.

The protein comprising the amino acid sequence of (a) or (b) above is a recombinant protein in which the amino acid sequence of the fission yeast Pvg1 protein is modified, and pyruvate is added to galactose located at the sugar chain end of the complex sugar chain. Has activity to transfer.
Among these recombinant proteins, a protein having an amino acid sequence represented by SEQ ID NOs: 1, 6, 8, 9, 10, 11, 13 is preferable from the viewpoint of high enzyme activity. A protein consisting of the amino acid sequence shown is more preferred.
According to the protein comprising the amino acid sequence of (a) or (b) above, a complex type sugar chain containing pyruvated galactose and a glycoprotein having the sugar chain can be obtained.

<Recombinant gene>
In addition, the protein comprising the amino acid sequence (a) or (b) may be translated from a recombinant gene comprising any one of the following base sequences (c) to (f) encoding the protein.
Therefore, the recombinant gene of the present invention is DNA comprising any one of the following base sequences (c) to (f).
(C) DNA consisting of the base sequence represented by SEQ ID NOs: 14 to 26,
(D) a protein consisting of a base sequence represented by SEQ ID NOs: 14 to 26, wherein one to several bases are deleted, substituted or added, and has a galactose-specific pyruvate transfer activity; DNA from fission yeast encoding,
(E) The homology (identity of the base sequence) with the base sequences represented by SEQ ID NOs: 14 to 26 is 80% or more, preferably 85% or more, more preferably 90% or more, and further preferably 95% or more. DNA derived from fission yeast, which consists of a base sequence and encodes a protein having galactose-specific pyruvate transfer activity, or
(F) a pyruvin specifically composed of a base sequence capable of hybridizing under stringent conditions with a DNA comprising a base sequence complementary to the DNA comprising the base sequence represented by SEQ ID NOs: 14 to 26 and under stringent conditions. A DNA derived from fission yeast encoding a protein having acid transfer activity.

  Here, the number of bases that may be deleted, substituted or added is preferably 1 to 30, more preferably 1 to 15, particularly preferably 1 to 10, and most preferably 1 to 5. .

  In the present invention and the present specification, “stringent conditions” includes, for example, a method described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Sambrook et al., Cold Spring Harbor Laboratory Press). For example, 5 × SSC (composition of 20 × SSC: 3M sodium chloride, 0.3M citric acid solution, pH 7.0), 0.1 wt% N-lauroyl sarcosine, 0.02 wt% SDS, 2 wt% The hybridization conditions can be exemplified by incubation for several hours to overnight at 55 to 70 ° C. in a hybridization buffer comprising 50% formamide and a blocking reagent for nucleic acid hybridization. The washing buffer used for washing after incubation is preferably a 0.1 × SSC solution containing 0.1 wt% SDS, more preferably a 0.1 × SSC solution containing 0.1 wt% SDS.

<Recombinant vector>
The recombinant vector of the present invention contains the above-described recombinant gene of the present invention. Specifically, the recombinant vector of the present invention comprises any one of the base sequences (c) to (f). It contains DNA.
The recombinant vector of the present invention is not particularly limited as long as it contains DNA comprising any one of the base sequences (c) to (f), and any expression vector (c) to (f) can be used. It may be obtained by inserting a DNA comprising such a base sequence. When inserting the DNA into an expression vector, an initiation codon ATG may be added to the 5 ′ end of the DNA, and as long as the addition of the pyruvate transfer activity is maintained in a galactose-specific manner, a histidine tag, etc. Sequences may be added to the 5 ′ or 3 ′ end to express the recombinant protein.

What is necessary is just to select suitably the well-known expression vector suitable for a host cell as said expression vector. Examples of Escherichia coli include ColE-type plasmids typified by pBR322 derivatives, pACYC-type plasmids having a p15A origin, pSC-type plasmids, and Bac-type F factor-derived mini-F plasmids. Other examples include expression vectors having tryptophan promoters such as trc and tac, lac promoter, T7 promoter, T5 promoter, T3 promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, tetracycline-inducible promoter, and the like.
Examples of the cell-free vector include an expression vector having a T7 promoter and an expression vector having a T3 promoter mentioned in the cell-based vector; a vector for wheat cell-free protein synthesis such as a pEU-based plasmid having an SP6 promoter or a T7 promoter. It is done.

  When yeast is used as a host, examples of preferable expression vectors include expression vectors such as YEp, YCp, and YIp. Expression vectors that can be used in other known yeasts can be selected. .

In addition, a cell-free vector may be used as the expression vector. In this case, in protein synthesis using a cell-free vector, first, cDNA is transcribed using a transcription system to synthesize mRNA. Examples of such transcription systems include conventionally known ones that are transcribed with RNA polymerase. Examples of the RNA polymerase include T7 RNA polymerase.
Next, using a cell-free protein synthesis system, which is a translation system, mRNA is translated and a protein is synthesized. This system contains elements necessary for translation, such as ribosomes, translation initiation factors, translation elongation factors, dissociation factors, and aminoacyl-tRNA synthetases. Examples of such protein translation systems include Escherichia coli extract, rabbit reticulocyte extract, and wheat germ extract.
Furthermore, there is a reconstituted cell-free protein synthesis system in which the elements necessary for the translation are composed solely of independently purified factors.
Protein synthesis using a cell-based vector will be described later in <Transformant>.
The recombinant protein of the present invention can be purified from a protein synthesized using a cell-based vector or a cell-free vector. Examples of the purification method include salting out methods and methods using various chromatographies. If the expression vector is designed to express a tag sequence such as a histidine tag at the N-terminus or C-terminus of the target protein, use an affinity column that uses a substance having an affinity for this tag, such as nickel or cobalt. A purification method is mentioned. In addition, the purity of the recombinant protein of the present invention can be increased by purifying it by appropriately combining ion exchange chromatography or gel filtration chromatography.

<Transformant>
The transformant of the present invention contains the recombinant vector of the present invention. As a method for obtaining the transformant of the present invention, the transformant may be obtained by introducing the recombinant vector of the present invention into a host. The transformant of the present invention preferably expresses the recombinant gene of the present invention. That is, “including a recombinant vector” may be a form in which a vector is present in a host cell or a form in which a vector-derived sequence is inserted into the host genome, and is inserted into the host genome. In this case, it is sufficient if a part of the vector containing the recombinant gene of the present invention is inserted into the host genome.

As the host used in the present invention, bacteria, fungi, animal cells, plant cells, and multicellular organisms other than humans composed of these cells can be used.
When obtaining a complex-type sugar chain or a human complex-type sugar chain, when using a host that does not have the ability to biosynthesize these complex-type sugar chains, the recombinant protein of the present invention is used as described above. It is preferably used for the purpose of obtaining from secretions in or outside the host cell.

  When using a host having the ability to biosynthesize human complex sugar chains, the recombinant protein of the present invention is used as described in <Method for producing sugar chains> or <Method for producing sugar chain-modified proteins> described later. Can be made to function in a host cell, and a human-type sugar chain or a human-type sugar chain-modified protein can be obtained. As such a host, in addition to cells derived from humans, bacteria, fungi, animal cells, plant cells, or multicellular organisms composed of these cells, which originally have no ability to biosynthesize human sugar chains, are previously human. A transformant into which a component of a biosynthetic system of type sugar chain is introduced may be used as a host.

  The method for introducing a recombinant vector into a cell is not particularly limited as long as it is a method for introducing a nucleic acid into the cell, and a conventionally known method can be used. Examples thereof include an electroporation method, a calcium phosphate method, an injection method, a particle gun method, and a method utilizing infection by a virus or bacteria.

<Method for producing pyruvate-containing complex sugar chain>
The method for producing a pyruvate-containing complex sugar chain of the present invention includes a step of transferring pyruvate to galactose located at the sugar chain end using the recombinant protein of the present invention or the transformant of the present invention. As described above, the recombinant protein can be obtained by translating the protein in a cell system or a cell-free system using the recombinant vector of the present invention. When the sugar chain is a human type sugar chain, production of a pyruvate-containing human complex type sugar chain is realized.
In the step of transferring pyruvic acid, the sugar or sugar chain to be pyruvated (acceptor substrate) is combined with the recombinant protein of the present invention or the transformant of the present invention in the presence of a pyruvic acid donor (donor substrate). It can be carried out by reacting.

  When the step of transferring pyruvic acid is performed by reacting with the recombinant protein of the present invention, the reaction is preferably performed in an aqueous solution, and the reaction conditions such as the temperature of the aqueous solution, the reaction time, the acceptor substrate concentration, the donor substrate concentration, etc. It is possible to appropriately select appropriate conditions in consideration of the type of substrate. As preferable reaction conditions, the pH is preferably in the range of pH 5.0 to 7.0, more preferably in the range of pH 5.5 to 6.5, and in the range of pH 5.5 to 6.0. More preferably. The reaction temperature is preferably in the range of 25 to 45 ° C, more preferably in the range of 30 to 40 ° C, and still more preferably in the range of 33 to 38 ° C. The reaction time is preferably in the range of 0.1 to 12 hours, more preferably in the range of 0.1 to 5 hours, and further preferably in the range of 0.1 to 1 hour. The acceptor substrate concentration is preferably in the range of 5-50 μmol / ml, more preferably in the range of 20-50 μmol / ml, and even more preferably in the range of 40-50 μmol / ml. The donor substrate concentration is also preferably in the range of 2 to 10 μmol / ml, more preferably in the range of 5 to 10 μmol / ml, and more preferably in the range of 8 to 10 μmol / ml. it can. The molar concentration ratio of the donor substrate to the acceptor substrate is preferably in the range of 0.1 to 10 times, more preferably in the range of 1 to 10 times, and further preferably in the range of 1 to 5 times. . The molar concentration ratio of the recombinant protein of the present invention to the donor substrate or acceptor substrate is preferably in the range of 1 to 10 times, more preferably in the range of 2 to 10 times, and in the range of 5 to 10 times. More preferably.

  When the step of transferring pyruvic acid is carried out by reacting with the transformant of the present invention, the reaction is carried out in the cells of the transformant, and the acceptor substrate and donor substrate present in the cells are transformed into the transformant. This is performed by reacting with the recombinant protein of the present invention translated in the converter. The reaction conditions are the same as those for preferred culture, breeding, etc. of the transformant, and preferred conditions may be selected according to the type of transformant.

<Method for producing pyruvic acid-containing complex sugar chain-modified protein>
The method for producing a pyruvate-containing complex sugar chain-modified protein of the present invention comprises a step of transferring pyruvic acid to galactose located at the sugar chain end using the recombinant protein of the present invention or the transformant of the present invention. .
In the step of transferring pyruvic acid, the sugar or sugar chain to be pyruvated (acceptor substrate) is combined with the recombinant protein of the present invention or the transformant of the present invention in the presence of a pyruvic acid donor (donor substrate). It can be carried out by reacting.
At this time, the acceptor substrate may be a glycoprotein having a complex type sugar chain or a complex type sugar chain. When the sugar chain of the acceptor substrate sugar chain or the glycoprotein is a human-type sugar chain, production of a pyruvate-containing human complex-type sugar chain is realized.

  When a complex type sugar chain is used as an acceptor substrate, the obtained pyruvate-containing complex type sugar chain-modified protein may be added to an arbitrary protein to produce a pyruvate-containing complex type sugar chain-modified protein. In this case, an enzyme having glycosyltransferase addition activity can be exemplified, and a specific endo-type glycosidase such as Endo-M can be exemplified as a specific enzyme.

  When a glycoprotein having a complex sugar chain is used as a substrate, pyruvic acid may be added to galactose contained in the sugar chain of the glycoprotein to produce a pyruvate-containing complex sugar chain-modified protein. .

Either the recombinant protein of the present invention or the transformant of the present invention may be used for the production of the pyruvate-containing complex sugar chain-modified protein of the present invention. As a preferable reaction condition when using the recombinant protein or the transformant of the present invention, it is preferable to select a method according to the above-described method for producing a pyruvic acid-containing complex sugar chain.
In the production of the pyruvic acid-containing complex-type glycosylated protein of the present invention, a transformant is more preferably used. This is from the viewpoint that a series of reactions such as sugar chain synthesis, pyruvate transfer, glycoprotein synthesis, and the like are performed in the transformant, and acquisition of the sugar chain-modified protein becomes very easy.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

<Production of recombinant gene, recombinant vector and transformant>
As a result of intensive studies, the inventors have estimated amino acids that may be involved in substrate recognition of Pvg1 protein in fission yeast. Based on this information, a recombinant gene DNA encoding the amino acid sequence shown in SEQ ID NOs: 1 to 13 was obtained. Among these, the recombinant gene obtained by changing histidine, which is the amino acid sequence shown in SEQ ID NO: 1 and which is the 168th amino acid sequence of the original fission yeast Pvg1 protein, to alanine is denoted as Pvg1H168A.

  Similarly, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 2 is Pvg1H168G, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 3 is Pvg1H168V, and the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 4 is Pvg1H168I. The recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 5 is Pvg1H168M, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 6 is Pvg1H168P, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 7 is Pvg1H168S, the sequence The recombinant gene encoding the amino acid sequence shown in No. 8 is shown in Pvg1H168T, the recombinant gene encoding the amino acid sequence shown in SEQ ID No. 9 is shown in Pvg1H168N, and SEQ ID No. 10 The recombinant gene encoding the amino acid sequence is Pvg1H168Q, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 11 is Pvg1H168C, the recombinant gene encoding the amino acid sequence shown in SEQ ID NO: 12 is Pvg1H168D, and the amino acid shown in SEQ ID NO: 13 The recombinant gene encoding the sequence is denoted Pvg1H168E.

These recombinant genes derived from Pvg1H were inserted into the pET32b-HRV3C vector according to a well-known method, and a total of 13 types of recombinant vectors containing the genes were prepared.
The obtained recombinant vector was transformed into Escherichia coli (BL21 strain) by an existing method, and Escherichia coli containing the recombinant vector was selected to obtain a total of 13 types of transformants.

<Production of recombinant protein>
A total of 13 types of recombinant proteins encoded by the above recombinant genes derived from Pvg1 were produced. A typical example is the case of the Pvg1H168A gene. The transformed Escherichia coli expressing the Pvg1H168A gene was cultured to mass-produce Pvg1H168A protein in the microbial cells. Subsequently, the transformed E. coli is cultured with shaking at 30 ° C. until the turbidity (OD 600 nm) reaches 0.5, and then IPTG is added to a final concentration of 0.1 mM, followed by shaking at 15 ° C. for 48 hours. went. The culture solution was centrifuged to obtain bacterial cells and suspended in a disruption buffer. The crushing buffer was prepared by adding 0.5 M MOPS-NaOH, pH 7.4; 0.1% Triton X-100; complete EDTA-free (manufactured by Roche) to 50 ml.
Then, the microbial cell was crushed using the ultrasonic crusher (UD-201, the product made by TOMY) on ice. The crushing condition is OUTPUT. After crushing for 10 seconds at Duty 50, this was repeated 9 times by standing on ice for 10 seconds. After crushing, the supernatant was obtained by centrifugation at 15000 rpm for 5 minutes.
Since the Pvg1H168A protein obtained by the above method has a HIS tag, purification was performed according to the protocol of HisTrap ™ FF 1 ml (GE healthcare) to obtain a purified Pvg1H168A protein.

<Transfer of pyruvic acid to galactose>
Next, the transfer activity of pyruvic acid to the galactose of the Pvg1H168A protein was confirmed. As a pyruvate donor substrate, phosphoenolpyruvate (PEP) was used. Gal-β1,4-GlcNAc-pNP was used as an acceptor substrate that undergoes pyruvate transfer. The reaction was performed under the following conditions.

[Example 1]
The reaction liquid was adjusted with the following composition. The reaction solution was adjusted with water to a total of 50 μL.
Reaction buffer: 1 M MOPS buffer (pH 6.0) 5 μL
Donor substrate: 50 mM PEP 5 μL
Acceptor substrate: 50 mM Gal-β1,4-GlcNAc-pNP
(Cosmo Bio, N501255) 5 μL
Pyruvate transferase: Pvg1H168A 10 μL
Reaction temperature: 30 ° C
Reaction time: 10-30 minutes

  In Reference Examples 1 and 2 and Comparative Example 1 below, wild-type Pvg1 protein of fission yeast was used as pyruvate transferase. Moreover, it reacted using the following substrates as an acceptor substrate. Other reaction conditions were the same as in Example 1.

[Reference Example 1] Acceptor substrate: 50 mM Gal-β-pNP (No 130555, manufactured by Seikagaku Corporation) 5 μL
[Reference Example 2] Acceptor substrate: 50 mM Gal-β1,4-Glc-pNP (Sigma N1752) 5 μL
[Comparative Example 1] Acceptor substrate: 50 mM Gal-β1,4-GlcNAc-pNP
5 μL
Table 1 shows the substrates and enzymes used in Example 1, Reference Examples 1 and 2, and Comparative Example 1.

Each reaction solution before and after the reaction was analyzed by high performance liquid chromatography (HPLC). The HPLC apparatus used was GL7410 (manufactured by GL Science, pump GL-7410, UV detector GL-7451), and COSMOSIL column (Nakarai test, Cosmosil, Code No. 39103-31, 5C18-P, 4.6 × 150 mm). ) Was used. The reaction results in Example 1 and Comparative Example 1 are shown in FIG. In FIG. 1, the peak of number 1 represents Gal-β1,4-GlcNAc-pNP, and the peak of number 2 represents PvGal-β1,4-GlcNAc-pNP. As is clear from the results shown in FIG. 1, Example 1 using Pvg1H168A as the pyruvate transferase is PvGal-β1,4 compared to Comparative Example 1 using wild type Pvg1 as the pyruvate transferase. The amount of -GlcNAc-pNP produced was greatly increased, and the increase was about 20 times the amount of PvGal-β1,4-GlcNAc-pNP produced in Comparative Example 1.
Table 1 shows the transfer activity of pyruvic acid for each acceptor substrate calculated from the results of the reactions of Example 1, Reference Examples 1 and 2 and Comparative Example 1. The transfer activity was calculated from the waveform area of the HPLC analysis result. The transfer activity represents a relative value when the value of Reference Example 1 is 100%.

  According to the comparison between Reference Example 2 and Comparative Example 1, the pyruvate transfer activity of the wild-type Pvg1 protein is significantly higher when the sugar bound to Gal in the acceptor substrate is GlcNAc than when GlcNAc is used. It turns out that it falls. However, as is clear from the results of Example 1, the pyruvate transfer activity of Pvg1H168A was able to achieve a high value even when the sugar bound to Gal in the acceptor substrate was GlcNAc. .

As described above, in addition to Pvg1H168A, Pvg1H168G, Pvg1H168V, Pvg1H168I, Pvg1H168M, Pvg1H168P, Pvg1H168S, Pvg1H168T, Pvg1H168N, Pvg1P168H, Pvg1H168Q
For comparison, histidine, which is the 168th amino acid sequence of the original fission yeast Pvg1 protein, was changed to leucine, phenylalanine, tryptophan, tyrosine, arginine, and lysine, respectively. Pvg1H168L, Pvg1H168F, Pvg1H168W, Pvg1H168Y, Vectors containing Pvg1H168R and Pvg1H168K were prepared, and Pvg1H168L, Pvg1H168F, Pvg1H168W, Pvg1H168Y, Pvg1H168R and Pvg1H168K proteins were obtained in the same manner as described above.
The transfer activity of pyruvic acid to Gal-β1,4-GlcNAc-pNP of each of these 19 recombinant proteins was measured. Table 2 shows the measurement results. The activity value is expressed as a relative value when the activity of wild-type Pvg1 is defined as 100%.

<Transfer of pyruvic acid to PA001>
Next, the transfer activity of pyruvate of the Pvg1H168A protein to galactose contained in the sugar chain of PA001 was verified.
[Example 2]
The reaction liquid was adjusted with the following composition. The reaction solution was adjusted with water to a total of 50 μL.
Reaction buffer: 1 M MOPS buffer (pH 6.0) 5 μL
Donor substrate: 10 mM PEP 1 μL
Acceptor substrate: 50 mM PA001 (Takara Bio Inc.) 3 μL
Pyruvate transferase: Pvg1H168A 50 μg
Reaction temperature: 30 ° C
Reaction time: 2 hours

  PA001 is a standard sugar chain reagent having the structure of a human complex type sugar chain, and has a pyridylamino (PA) -terminated PA terminal. The sugar chain structure of PA001 is shown in FIG.

  In Comparative Example 2 below, fission yeast wild-type Pvg1 protein was used as pyruvate transferase, PA001 was used as an acceptor substrate, and the other conditions were the same as in Example 2.

  Table 3 shows the substrates and enzymes used in Example 2 and Comparative Example 2.

Each reaction solution before and after the reaction was analyzed in the same manner as in Example 1.

The reaction results in Example 2 and Comparative Example 2 are shown in FIGS. 3 and 4, the peak of number 3 represents PA001, the peak of number 4 is presumed that one pyruvate has been transferred to PA001 (Pv), and represents the peak of PA001, and the peak of number 5 represents pyruvate to PA001. Represents two transferred (Pv) ₂ PA001. As is clear from the data in FIG. 3, in Comparative Example 2 using wild-type Pvg1 as pyruvate transferase, pyruvate cannot be transferred to PA001 or the pyruvate transferase activity is extremely low. In Example 3 in which Pvg1H168A was used as the pyruvate transferase shown in FIG. 4, a prominent peak that can be presumed that PA001 was pyruvated was observed.

(Confirmation of pyruvin oxidation (1))

PvGal has the property that pyruvic acid is removed under weak acid conditions at 100 ° C. Utilizing this property, it was verified by the following reaction conditions whether the substance having the peak No. 5 obtained in Example 2 was oxidized by pyruvin.

Reaction temperature: 100 ° C

Reaction time: 6 hours

Reaction solution: 0.02M HCl,

Substrate: the eluate of the peak 5 obtained by purifying the reaction solution obtained in Example 2

The analysis result by HPLC of the reaction solution after the above weak acid and 100 ° C. condition treatment is shown in FIG. For control, the results when water is used in the reaction solution instead of 0.02M HCl are also shown. As can be seen from the results of FIG. 5, the peak of No. 5 in FIG. 5 is not observed after treatment with the weak acid at 100 ° C., and the peak of PA001 of No. 1 that is not pyruvated (the same position as the peak of No. 3). Back to the peak. This strongly suggested that the substance indicated by the peak of No. 5 in FIG. 5 was pyruvic oxidized PA001.

(Confirmation of pyruvin oxidation (2))

Moreover, PvGal has the property that it is not degraded by β-galactosidase. Utilizing this property, it was verified by the following reaction conditions whether the substances having the peaks of Nos. 3 to 5 obtained in Example 2 were oxidized by pyruvin.

Reaction temperature: 37 ° C

Reaction time: 12 hours

Enzyme: β-galactosidase (Wako Pure Chemicals) 10U

Substrate: reaction solution obtained in Example 2

The analysis result by HPLC with respect to the reaction liquid after β-galactosidase treatment obtained by the reaction as described above is shown in FIG. From this result, it can be seen that only the peak of number 5 in FIG. 6 has no peak shift. This indicates that galactose in the molecule is pyruvated. Moreover, it is considered that the peaks of No. 3 and No. 4 in FIG. 6 are shifted to No. 3 ′ and No. 4 ′, respectively. Since the shift width of the No. 4 peak is smaller than the shift width of the No. 3 peak, it is considered that the galactose in the molecule indicated by the No. 4 peak is partially pyruvated. Therefore, it was confirmed that the substance indicated by the peaks of No. 4 and No. 5 in FIG. 6 is pyruvic oxidized PA001.

The configurations and combinations thereof in the embodiments described above are examples, and the addition, omission, replacement, and other modifications of the configurations can be made without departing from the spirit of the present invention. Further, the present invention is not limited by each embodiment, and is limited only by the scope of the claims.

<Characteristic analysis of pyruvate glycopeptide by lectin array analysis>
Next, the pyruvic oxidized glycopeptide was characterized by lectin array analysis.
[Example 3] Sialic acid was removed from a sialylglycopeptide having the following chemical formula (manufactured by Fushimi Pharmaceutical Co., Ltd.) to prepare an asialoglycopeptide, which was used to prepare a pyruvin glycopeptide in a reaction solution having the following composition. .
Reaction buffer: 0.2M MOPS buffer (pH 7.5)
Donor substrate: 40 mM phosphoenolpyruvate Acceptor substrate: 1 mg Asialoglycopeptide pyruvate transferase: Pvg1 mutant 0.5 mg
Reaction temperature: 30 ° C
Reaction time: 16 hours

  Next, the sialyl glycopeptide (SGP) and the pyruvine oxidized glycopeptide (PVGP) synthesized above were labeled with Cy3 which is a fluorescent substance.

  A lectin array having 96 types of sugar specificity specified in FIGS. 7 and 8 (96 types of lectins are recombinants produced by 38 types of E. coli, etc., but other types of lectins are commercially available and Purified from plants, etc. For the origin of lectin, Tateno et al, J. Biol. Chem., 286, 20345-20353 (2011) and Hirabayashi et al, Electrophoresis, 32, 1118-1128 (2011) Etc.) and the fluorescent glycopeptide labeled above was added and incubated at 20 ° C. overnight. Wash the array with probing buffer (25 mM Tris-HCl, pH 7.5, 140 mM NaCl (TBS) buffer containing 2.7 mM KCl, 1 mM CaCl2, 1 mM MnCl2, 1% Triton X-100) The fluorescence was measured with GlycoStation Reader 1200 (manufactured by Glycotechnica) (FIGS. 9A and 9B). The obtained raw data was digitized and graphed (FIGS. 10A, 10B, 11A, and 11B).

(result)
The above analysis revealed the following.
(1) The pyruvic oxidized glycopeptide interacts with lectins that recognize α2,6-linked terminal sialic acids such as SNA, SSA, TJAI, rPSL1a.
(2) The pyruvic oxidized glycopeptide does not interact with lectins that recognize α2,3-linked sialic acid such as MAL, MAH, ACG, rACG, and rGal8N.
(3) These results show more clearly that pyruvic oxidized galactose is similar to the structure of sialic acid bound α2,6 to galactose (see FIG. 12).

  The pyruvic acid-containing complex sugar chain of the present invention is applicable to any glycoprotein pharmaceutical having galactose. In addition, by controlling the pyruvic acid content degree, there is a possibility that a useful pharmaceutical whose duration of action is appropriately controlled can be provided.

Claims (17)

The following (a) or (b), a recombinant protein:
(A) a protein comprising the amino acid sequence represented by any one of SEQ ID NOs: 1 to 13;
(B) consisting of an amino acid sequence in which one to several amino acids are deleted, inserted, substituted or added at a site other than amino acid number 168 of the amino acid sequence shown in any one of SEQ ID NOs: 1 to 13; Protein with pyruvate transfer activity specific to galactose.   A recombinant gene encoding the protein according to claim 1.   A recombinant vector comprising the gene according to claim 2.   A transformant comprising the recombinant vector according to claim 3.   A method for producing a pyruvic acid-containing complex type sugar chain, comprising the step of transferring pyruvic acid to galactose located at the sugar chain end using the recombinant protein according to claim 1.   A method for producing a pyruvic acid-containing complex type sugar chain, comprising a step of transferring pyruvic acid to galactose located at the sugar chain end using the transformant according to claim 4.   The method for producing a pyruvic acid-containing complex sugar chain according to claim 5 or 6, wherein the sugar bonded to galactose located at the sugar chain terminal is N-acetylglucosamine.   The method for producing a pyruvic acid-containing complex type sugar chain according to any one of claims 5 to 7, wherein the sugar chain terminal is a human type sugar chain.   A method for producing a pyruvic acid-containing complex sugar chain-modified protein, comprising the step of transferring pyruvic acid to galactose located at the end of a sugar chain using the recombinant protein according to claim 1.   The manufacturing method of pyruvic acid containing complex type | mold sugar chain modification protein which has the process of transferring pyruvic acid to the galactose located in the terminal of sugar chain using the transformant of Claim 4.   The method for producing a pyruvic acid-containing complex type sugar chain-modified protein according to claim 9 or 10, wherein the sugar bonded to the protein side of galactose located at the sugar chain terminal is N-acetylglucosamine.   The method for producing a pyruvate-containing complex type sugar chain-modified protein according to any one of claims 9 to 11, wherein the sugar chain terminal is a human type sugar chain.   A pyruvic acid-containing complex type sugar chain comprising a sugar chain structure provided with pyruvic oxidized galactose formed by binding pyruvic acid to galactose, wherein the sugar chain structure has a basic structure of an asparagine-linked sugar chain.   The pyruvic acid-containing complex type sugar chain according to claim 13, wherein the sugar chain structure is a human type sugar chain.   The pyruvic acid-containing complex sugar chain according to claim 14, wherein the pyruvic oxidized galactose is located at a sugar chain terminal of the human sugar chain.   The pyruvic acid-containing complex type sugar chain according to any one of claims 13 to 15, wherein the sugar bonded to the pyruvic oxidized galactose is N-acetylglucosamine.   A pyruvic acid-containing complex sugar chain-modified protein having the pyruvic acid-containing complex sugar chain according to any one of claims 13 to 16.