ES2365388T3 - Chondroitin-Polymerase and DNA that encodes it. - Google Patents

Abstract

Isolated protein selected from the group consisting of the following sections (A) and (B): (A) a protein comprising the amino acid sequence represented by the SEC. ID. No. 2; (B) a protein comprising the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity.

Description

Background of the present invention

one.

Field of the present invention

The present invention relates to a new chondroitin polymerase (chondroitin synthase), to a DNA encoding it, to a process for producing chondroitin polymerase, to a process for producing a sugar chain containing the chondroitin disaccharide unit repetitive, to a hybridization probe for chondroitin polymerase and the like.

2.

Brief description of the prior art

First, the abbreviations frequently used herein are described.

In the formulas and the like, "GlcUA", "GalNAc", "GlcNAc", "UDP" and "-" represent D-glucuronic acid, N-acetyl-Dgalactosamine; N-acetyl-D-glucosamine; uridine 5'-diphosphate and a glycosidic bond, respectively.

Chondroitin is a sugar chain comprising a repetitive disacarid structure of the GlcUA moiety and the GalNAc moiety (-GlcUAβ (1-3) -GalNAcβ (1-4)); Also referred to hereinafter as "chondroitin structure") and a sugar chain in which the most sulfated chondroitin is chondroitin sulfate.

As for an enzyme that synthesizes chondroitin from a GlcUA donor and a GalNAc donor by transferring GlcUA and GalNAc alternately to an acceptor (chondroitin polymerase or chondroitin synthase) and DNA encoding it, only one chondroitin is known. Pasteurella multocida synthase (J. Biol. Chem., 215 (31), 24124-24129 (2000)).

In addition, certain strains of Escherichia coli (serotype 05: K4 (L): H4 of Escherichia coli, hereinafter referred to as "K4 strain of Escherichia coli") produce a polysaccharide with chondroitin structure, as a capsular antigen, but its structure is a repetitive trisacaridic structure in which the fructose is attached to a side chain of the GlcUA moiety in the β2-3 position. Therefore, it was not clear whether Escherichia coli strain K4 actually has a chondroitin polymerase as its own capsular antigen synthesis system.

Summary of the invention

An objective of the present invention is to provide a new chondroitin polymerase, a DNA encoding it, a process for producing chondroitin polymerase, a method for producing the sugar chain containing the repetitive polysaccharide chondroitin unit, a probe of hybridization for chondroitin polymerase and the like.

This and other objectives of the present invention are achieved by a new chondroitin polymerase with the specific properties described below.

Brief description of the drawings

Figure 1 shows a restriction mapping of the phage clones λ containing a part of the R-I zone or the R-III zone of the K antigen gene group of the K4 strain of Escherichia coli.

Figure 2 shows the open reading frame (ORF) of the R-II region of the K antigen gene group of the K4 strain of Escherichia coli.

Figure 3 is a graph showing the transfer of GalNAc to the hexasaccharide of chondroitin C sulfate by the enzyme of the present invention.

Figure 4 is a graph showing the transfer of GalNAc to the hexasaccharide of chondroitin C sulfate by the enzyme of the present invention and the dimensions of the sugar chain produced.

Figure 5 is a graph showing the transfer of each monosaccharide when it was used as a donor UDP-GlcUA, UDP-GlcNAc or UDP-GalNAc, and was used as a hexasaccharide or heptasaccharide acceptor of chondroitin C sulfate.

Figure 6 is a graph showing the influence of temperature on the enzyme activity of the present invention.

Figure 7 is a graph showing the gel filtration models of the products of the enzymatic reaction after several periods of enzymatic reaction.

Figure 8 is a graph showing the relationship between the enzymatic reaction period and the amount of radioactivity incorporation.

Figure 9 is a graph showing the relationship between the incorporated radioactivity (V) and the concentration in the UDP-sugar substrate (S).

Figure 10 is a graph presenting double reciprocal representations.

Detailed description of the invention

The present inventors have carried out intensive studies and have discovered a new chondroitin polymerase produced by specific microorganisms (Escherichia coli strain 4 (serotype 05: K4 (L): Escherichia coli H4, ATCC 23502)), isolated cDNA encoding chondroitin polymerase, and has managed to prepare chondroitin polymerase using cDNA. Thus, the present invention has been realized.

In addition, this and other objects of the present invention have been achieved by a method of producing chondroitin polymerase by isolating the cDNA encoding chondroitin polymerase and using the cDNA. The term "chondroitin synthesis" as used herein is a concept that includes lengthening the chondroitin sugar chain by transferring and adding monosaccharides to a sugar chain such as chondroitin. Therefore, the reaction to lengthen the chondroitin sugar chain by transferring alternately and adding the monosaccharides (GlcUA and GalNAc) to the sugar chain to synthesize chondroitin is included in a concept of "chondroitin synthesis".

A chondroitin polymerase (hereinafter also referred to as "enzyme of the present invention") having the following properties is described herein:

(one)

Action:

the polymerase transfers GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain of a GlcUA donor and a GalNAc donor, respectively;

(2)

Substrate Specificity:

the polymerase transfers GlcUA to an oligosaccharide that has GalNAc in its non-reduced terminal and a chondroitin structure of a GlcUA donor, but does not substantially transfer GalNAc to the oligosaccharide of a GalNAc donor;

the polymerase transfers GalNAc to an oligosaccharide that has GlcUA in its non-reduced terminal and a chondroitin structure of a GalNAc donor, but does not substantially transfer GlcUA to the oligosaccharide of a GlcUA donor;

(3) Influence of metal ions and the like:

The polymerase acts in the presence of Mn2 + ion but does not act substantially in the presence of Ca2 + ion, Cu2 + ion

or ethylenediaminetetraacetic acid.

The enzyme described herein is preferably derived from Escherichia coli.

The present invention relates to a protein selected from the group consisting of the following sections (A) and (B) (hereinafter referred to as "protein of the present invention"):

(TO)

a protein comprising the amino acid sequence represented by SEC. ID. No. 2;

(B)

a protein that comprises the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity.

The present invention relates to a DNA comprising one of the following sections (a) to (c) (hereinafter referred to as "DNA of the present invention"):

(to)

DNA encoding a protein consisting of the amino acid sequence represented by SEC. ID. No. 2;

(b)

DNA encoding a protein consisting of the amino acid sequence in which 1 to 30 residue (s) of

amino acids in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity;

(C)

DNA that hybridizes with

(i)

the DNA in (a), or

(ii)

DNA complementary to DNA in (a) under severe conditions in which hybridization is performed at 42 ° C in a solution containing 50% formamide, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's solution and 100 µg / ml salmon sperm DNA, and encoding a protein that has chondroitin polymerase activity.

The DNA in section (a) is preferably represented by SEC. ID. No. 1

The present invention relates to a vector comprising the DNA of the present invention (hereinafter referred to as "the vector of the present invention").

The vector of the present invention is preferably an expression vector.

The present invention relates to a transformant in which a host is transformed with the vector of the present invention (hereinafter also referred to as "transformant of the present invention").

The present invention relates to a process for producing a chondroitin polymerase, comprising: culturing the transformant of the present invention; and collecting chondroitin polymerase from the cultured material (hereinafter referred to as the "enzyme production process of the present invention").

The present invention relates to an agent for the synthesis of the sugar chain, which comprises the protein of the present invention and which has chondroitin polymerase activity.

The present invention relates to a process for producing a sugar chain represented by the formula

(3) below, comprising at least one step that allows the synthesizing agent of the present invention to contact a GalNAc donor and a sugar chain represented by the following formula (1) (hereinafter referred to as "method 1 of production of the sugar chain of the present invention "):

GlcUA-X-R1 (1) GalNAc-GlcUA-X-R1 (3)

in which X represents GalNAc or GlcNAc; R1 represents any group; and the other symbols have the same meanings described above.

The present invention relates to a process for producing a sugar chain represented by the formula

(4) below, comprising at least one step that allows the synthesizing agent of the present invention to contact a GlcNAc donor and a sugar chain represented by the following formula (1) (hereinafter referred to as "method 2" of production of the sugar chain of the present invention "):

GlcUA-X-R1 (1) GlcNAc-GlcUA-X-R1 (4)

in which all symbols have the same meanings described above.

The present invention relates to a process for producing a sugar chain represented by the formula

(5) below, comprising at least one step that allows the synthesizing agent of the present invention to contact a GlcUA donor and a sugar chain represented by the following formula (2) (hereinafter referred to as "method 3 of production of the sugar chain of the present invention "):

GalNAc-GlcUA-R2 (2) GlcUA-GalNAc-GlcUA-R2 (5)

in which R2 represents any group; and the other symbols have the same meanings described above.

The present invention relates to a process for producing a sugar chain represented by the formula

(6) and (8) below, comprising at least one step that allows the synthesizing agent of the present invention to contact a GalNAc donor a GlcUA donor and a sugar chain represented by the following formula (1) (hereinafter referred to as "process 4 of producing the sugar chain of the present invention"):

GlcUA-X-R1 (1) (GlcUA-GalNAc) n-GlcUA-X-R1 (6) GalNAc- (GlcUA-GalNAc) n-GlcUA-X-R1 (8)

in which n is an integer of 1 or more, and the other symbols have the same meanings described above.

The present invention relates to a process for producing a sugar chain represented by the following formulas (7) and (9), which comprises at least one step that allows the synthesizing agent of the present invention to contact a donor of GalNAc, a donor of GlcUA and a sugar chain represented by the following formula (2) (hereinafter referred to as "method 5 of producing the sugar chain of the present invention"):

GalNAc-GlcUA-R2 (2) (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (7) GlcUA- (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (9)

in which all symbols have the same meanings described above.

The present invention relates to a hybridization probe comprising a nucleotide sequence complementary to the nucleotide sequence represented by the SEC. ID. No. 1 (also referred to in the following as "probe of the present invention").

The present invention relates to a glycosyl transfer catalyst (hereinafter referred to as "catalyst of the present invention") comprising the protein of the present invention and having chondroitin polymerase activity.

The present invention is explained in more detail below.

<1> Enzyme of the present invention and protein of the present invention.

The enzyme described herein is a chondroitin polymerase that has the following properties (1) to (3).

(1) Action:

The enzyme described herein transfers GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain of a GlcUA donor and a GalNAc donor, respectively.

As a GlcUA donor, a nucleoside diphosphate-GlcUA is preferred, and in particular UDP-GlcUA is preferred. In addition, as a donor of GalNAc, a nucleoside diphosphate-GalNAc is preferred, and in particular UDP-GalNAc is preferred.

The enzyme described herein transfers GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain (acceptor) of these respective saccharide donors. For example, when GlcUA is first transferred to a non-reduced terminal of a sugar chain (acceptor), the monosaccharides are transferred in such a way that GalNAc is then transferred, then GlcUA is transferred, then GalNAc is transferred and so on. In the same way, when GalNAc is first transferred to a non-reduced terminal of a sugar chain (acceptor), the monosaccharides are transferred in such a way that GlcUA is then transferred, then GalNAc is transferred, then transferred GlcUA and so on. As a result, a repetitive disaccharide structure of GlcUA moiety and GalNAc moiety, ie a chondroitin skeleton, is synthesized by the enzyme of the present invention.

As a sugar chain that becomes a monosaccharide acceptor, a sugar chain having a chondroitin skeleton is preferable. As a sugar chain having a chondroitin skeleton, examples of chondroitin sulfate and chondroitin can be put as examples. Among chondroitin sulfates, a chondroitin sulfate is preferable which is mainly composed of a chondroitin 6-sulfate skeleton and also contains a small amount of a 4-sulfate-chondroitin structure (hereinafter referred to as "chondroitin sulfate C" ").

In addition, the sugar chain that becomes an acceptor is more preferably an oligosaccharide. The size of the oligosaccharide is not specifically restricted, but when the acceptor is a chondroitin sulfate C oligosaccharide, hexasaccharide or heptasaccharide is preferable, and tetrasaccharide or hexasaccharide is preferable when it is a chondroitin oligosaccharide.

In addition, it is preferable that the enzyme described herein is capable of transferring more GalNAc to a sugar chain having a hyaluronic acid structure (a repetitive disaccharide structure of the GlcUA moiety and the GlcNAc moiety) from a GalNAc donor . It is preferable that the sugar chain having a hyaluronic acid structure is also an oligosaccharide. The size of the oligosaccharide is not particularly restricted, but those that are composed of approximately hexasaccharides are particularly preferable.

(2) Substrate specificity:

The enzyme described herein transfers GlcUA to an oligosaccharide that has GalNAc at its non-reduced terminal and a chondroitin structure from a GlcUA donor, but does not substantially transfer GalNAc to the oligosaccharide from a GalNAc donor.

The enzyme described herein transfers GalNAc to an oligosaccharide having GlcUA in its non-reduced terminal and a chondroitin skeleton from a GalNAc donor, but does not substantially transfer GlcUA to the oligosaccharide from a GlcUA donor.

It is preferable that the enzyme described herein also does not substantially transfer GlcNAc from a donor of GlcNAc to an oligosaccharide that has GalNAc in its non-reduced terminal and also has a chondroitin skeleton. In addition, it is preferable that the enzyme described herein transfers more GlcNAc from a GlcNAc donor to an oligosaccharide that has GlcUA in its non-reduced terminal and also has a chondroitin skeleton. However, it is preferable that GlcUA is not substantially transferred from a GlcUA donor to an oligosaccharide produced by the transfer of GlcNAc.

In addition, it is preferable that the enzyme described herein transfers GlcNAc from a GlcNAc donor to an oligosaccharide that has GlcUA in its non-reduced terminal and that also has a hyaluronic acid backbone, but does not substantially transfer GlcUA from a GlcUA donor .

(3) Influence of metal ions and the like:

The enzyme described herein acts in the presence of Mn2 + ion but does not act substantially in the presence of Ca2 + ion, Cu2 + ion or ethylenediaminetetraacetic acid.

In addition, it is preferable that the enzyme described herein also acts in the presence of the Fe2 + or Mg2 + ion. On the other hand, it is preferable that the degree of action (enzymatic activity) of the enzyme described herein in the presence of Mn2 + ion is greater than its degree of action (enzymatic activity) in the presence of Fe2 + or Mg2 + ion.

In addition, it was observed that when a reaction is performed using the enzyme described herein at a temperature of 25 ° C or more, the size of the reaction product (chondroitin chain) becomes small as the reaction temperature increases (see , the examples shown below). Accordingly, the enzyme activity of the enzyme described herein is considered to decrease as the reaction temperature increases to 25 ° C or more under the reaction conditions described in the following examples.

It is preferable that the enzyme described herein comes from Escherichia coli. In particular, the Escherichia coli strain having a gene related to the production of the capsular polysaccharide is preferable, and the Escherichia coli strain, whose capsular antigen (K) is "K4", is more preferable.

As the Escherichia coli strain whose capsular antigen serotype is "K4", one can preferably cite Escherichia coli strain K4 (serotype 05: K4 (L): Escherichia coli B4), and more specifically, ATCC 23502 may be cited preferably , NCDC U1-41, number 2616 of the Freiburg collection and the like.

The enzyme of the present invention is a protein selected from the following sections (A) and (B):

(TO)

a protein comprising the amino acid sequence represented by SEC. ID. No. 2;

(B)

a protein that comprises the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity.

Although the mutation such as substitution, elimination, insertion, transposition and the like may occur in the amino acid sequences of the existing proteins in nature produced by the modification reactions within the cells or during protein purification after their formation, In addition to the polymorphism and mutation of the DNA molecules that encode them, it is known that certain proteins with said mutations have physiological and biological activities that are substantially identical to the corresponding proteins that do not have mutations. Such proteins that have slight structural differences but no significant differences in their functions are also described herein. It is the same case in which the previous mutation is artificially introduced into the amino acid sequence of the protein. In that case, it is possible to prepare larger varieties of mutants. For example, it is known that a polypeptide prepared by replacing a cysteine residue in the amino acid sequence of human interleukin 2 (IL-2) with a serine residue maintains the activity of interleukin 2 (Science, 224, 1431 (1984) ). In addition, it is known that a certain protein has a peptide zone that is not essential for its activity. For example, a signal peptide that exists in a protein that is secreted in the extracellular moiety and a pro-sequence that can be found in a protease precursor or the like corresponds to this case, and most of these areas are removed after translation. of proteins or during their conversion into active proteins. These proteins are proteins that are present in different ways from the point of view of their primary structure but ultimately have similar functions.

The expression "a few amino acid residues" as used herein means the number of amino acids that can produce mutation to such a degree that chondroitin polymerase activity is not lost, and in the case of the protein composed of 600 amino acid residues for example, means the number of about 2 to 30, preferably 2 to 15, and more preferably 2 to 8.

In addition, the protein of the present invention may contain an amino acid sequence of another protein or peptide, as long as it contains the amino acid sequence of section (A) or (B) above. That is, the protein of the present invention can be a fusion protein with another protein or peptide.

For example, fusion proteins of a protein comprising the amino acid sequence described in section (A) or (B) above with a marker peptide and the like are also included in the protein of the present invention. Said proteins of the present invention have the advantage that their purification can be easily accomplished. Examples of the above marker peptide include protein A, the insulin signal sequence, His, FLAG, CBP (calmodulin binding protein), GST (glutathione S-transferase) and the like. For example, its fusion protein with protein A can be conveniently purified by affinity chromatography using a solid phase bound to IgG. In the same way, a solid phase to which magnetic nickel binds can be used for a fusion protein with the His tag, and a solid phase to which an anti-FLAG antibody is bound can be used for a fusion protein with FLAG In addition, since a fusion protein with an insulin signal is secreted in the extracellular moiety (a medium or similar), extraction operations such as cell disintegration and the like become unnecessary. It is preferable that the protein of the present invention (the enzyme of the present invention) be soluble.

Preferred examples include a fusion protein with a peptide (His tag) represented by the amino acid sequence represented by the SEC. ID. No. 11. It is preferable to fuse this His tag continuously in a position just before the amino acid sequence represented by SEC. ID. No. 2. The fusion protein can be produced by expressing a DNA in which the nucleotide sequence represented by the SEC. ID. No. 4 is continuously connected to a position just before the nucleotide sequence represented by the SEC. ID. No. 1. The fusion protein is soluble.

"Chondroitin polymerase activity" can be detected according to an analytical method of glucosyltransferase generally used. Specifically, it can be detected as an activity to synthesize transferring GlcUA and GalNAc alternately to a non-reduced terminal of a sugar chain (acceptor), using a GlcUA donor, a GalNAc donor and a sugar chain that becomes the acceptor.

For example, when GlcUA is transferred from a GlcUA donor to a sugar chain that has GalNAc in its reduced terminal, it is transferred from a GalNAc donor to a sugar chain that has a GlcUA in its non-reduced terminal, it can be interpreted that It has activity to transfer GlcUa and GalNAc alternately to a non-reduced terminal of the sugar chain, namely chondroitin polymerase activity. It is preferable to use the method of measuring enzymatic activity described in the following Examples. Using said method, the elimination, substitution, insertion or transposition of amino acids that retain chondroitin polymerase activity can be easily selected.

The processes for the production of the enzyme of the present invention and of the protein of the present invention are not particularly restricted, and can be produced by expressing the DNA of the present invention described below in appropriate cells. In addition, those that are isolated from natural resources and produced by chemical synthesis and the like are included in the enzyme of the present invention and in the protein of the present invention as commonplace. The methods of producing the enzyme of the present invention (the protein of the present invention) which use in DNA of the present invention will be described below.

<2> DNA of the present invention

(to)

DNA encoding a protein consisting of the amino acid sequence represented by SEC. ID. No. 2;

(b)

DNA encoding a protein consisting of the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or

transpose, and that it has chondroitin polymerase activity;

(C)

DNA that hybridizes with

(i)

the DNA in (a), or

(ii)

DNA complementary to DNA in (a) under severe conditions in which hybridization is performed at 42 ° C in a solution containing 50% formamide, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's solution and 100 µg / ml salmon sperm DNA, and encoding a protein that has chondroitin polymerase activity.

The DNA is preferably represented by the SEC. ID. No. 1

"Severe conditions" as used herein means the conditions under which a so-called specific hybrid is formed but a non-specific hybrid is not formed (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press (1989) and the like). Examples of "severe conditions" include the conditions under which hybridization is carried out at 42 ° C in a solution containing 50% formamide, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x Denhardt's solution and 100 µg / ml salmon sperm DNA, and the product is washed with 2 x SSC, 0.1% SDS solution at room temperature and then with 0.1 x SSC, 0.1% SDS solution at 50 ° C.

The DNA of the present invention is generally obtained from the Escherichia coli strain having the K4 antigen, but DNA samples obtained from other organism species transformed and produced by chemical synthesis and the like are also included herein as something common The methods for producing the DNA of the present invention are also not specifically limited, but it is preferable to use, for example, the procedure described in the following examples.

Those skilled in the art readily understand that DNAs that have several different nucleotide sequences due to degeneracy of the genetic code are present as DNA of the present invention.

<3> Vector of the present invention

The vector of the present invention is a vector comprising the DNA of the present invention. Preferably the DNA in the vector of the present invention is the same as described in section <2> above. Furthermore, since the vector of the present invention is preferably used in the enzyme production process of the present invention which will be described later, it is preferably an expression vector.

The vector of the present invention can be prepared by inserting the DNA of the present invention into a known vector.

As the vector in which the DNA of the present invention is inserted, for example, an appropriate vector can be used that can express the introduced DNA (a phage vector, a plasmid vector or the like) and can optionally be selected in response to each cell host in which the vector of the present invention is inserted. Examples of the host vector system include a combination of a mammalian cell such as COS cell, 3LL-HK46 or the like with an expression vector for mammalian cell such as pGIR201 (Kitagawa, H. and Paulson, JC, J. Biol. Chem., 269, 1394-1401 (1994)), pEF-BOS (Mizushima, S. and Nagata, S., Nucleic Acid Res., 18, 5322 (1990)), pCXN2 (Niwa, H., Yamamura, K . and Miyazaki, J., Gene, 108, 193-200 (1991)), pCMV-2 (prepared by Eastman Kodak), pCEV18, pME18S (Maruyama et al., Med. Immunol., 20, 27 (1990)) , pSVL (prepared by Pharmacia Biotech) or the like and a combination of Escherichia coli with an expression vector for prokaryotic cells such as pTrcHis (manufactured by Invitrogen), pGEX (prepared by Pharmacia Biotech), pTrc99 (prepared by Pharmacia Biotech), pKK233 -3 (prepared by Pharmacia Biotech), pEZZZ18 (prepared by Pharmacia Biotech), pCH110 (prepared by Pharmacia Biotech), pET (prepared by Stratagene), pBAD (prepared by Invitrogen), pR SET (prepared by Invitrogen), pSE420 (prepared by Invitrogen) or the like. In addition, an insect cell, yeast, Bacillus subtilis and the like can also serve as an example as the host cell and several vectors corresponding thereto. Among the above host vector systems, a combination of Escherichia coli with pTrcHis is preferable.

In addition, as the vector into which the DNA of the present invention is inserted, a vector constructed in such a manner that expresses a fusion protein of the protein of the present invention (enzyme of the present invention) with a marker peptide, It can also be used, which, as described in section <1> above, is particularly preferable when the chondroitin polymerase expressed using the vector of the present invention is purified. Specifically, a vector comprising a nucleotide sequence expressing His (for example, the nucleotide sequence represented by SEQ ID NO. 4) is preferable.

When any of the above vectors is used, the DNA of the present invention may be connected to the vector after treating both with restriction enzymes and the like and optionally carrying out the soft termination and connection of a cohesive terminal so that the connection of the DNA of the present invention with the vector becomes possible.

As the production method of the vector of the present invention it can be used and it is preferable, for example, the procedure described in the following examples.

<4> Transformant of the present invention

The transformant of the present invention is a transformant in which a host is transformed with the vector of the present invention.

The "host" as used herein may be any host in which recombination can be performed by the vector of the present invention but one that can exert the function of the DNA of the present invention or a recombinant vector is preferable. which the DNA of the present invention is inserted. Examples of host include animal cells, plant cells and microbial cells, and mammalian cells such as COS cells (COS-1 cell, CO-7 cells and the like), 3LLHK46 cell, etc., Escherichia coli, cells are included. of insect, yeast, Bacillus subtilis and the like. The host may optionally be selected in response to each vector of the present invention, but, for example, when using a vector of the present invention prepared based on pTrcHis, it is preferable to select the Escherichia coli strain.

The host can be transformed by the vector of the present invention in the usual manner. For example, the host can be transformed by introducing the vector of the present invention into the host by a procedure using a commercially available transfection reagent, a procedure with DEAEdextran, electroporation or the like.

The transformant of the present invention obtained in this way can be used in the enzymatic production process of the present invention described below.

<5> Enzymatic production process of the present invention

The enzymatic production process of the present invention is a process for producing a chondroitin polymerase, which comprises cultivating the transformant of the present invention; and collect a chondroitin polymerase from the culture material.

The term "culture" as used herein means a general idea that includes the cultivation of cells or microorganisms as the transformant of the present invention itself and the cultivation of an animal, insect or the like in which the cells as the transformant of the present invention. In addition, the term "culture material" as used herein means a concept that includes a medium (culture medium supernatant) and host cells cultured after cultivation of the transformant of the present invention, the secreted matter, the excreted matter and the like.

The culture conditions (medium, culture condition and the like) are appropriately selected based on the host to be used.

According to the enzymatic production process of the present invention, various forms of chondroitin polymerase can be produced, based on the transformant to be used.

For example, when a transformant transformed with an expression vector constructed to express a fusion protein with a marker peptide is cultured, a chondroitin polymerase fused to the marker peptide is produced. Specifically, for example, a chondroitin polymerase fused to the His tag is produced to grow a transformant transformed with an expression vector constructed to effect the expression of a protein in which the amino acid sequence represented by SEC. ID. No. 11 is continuously fused to a position just before the amino acid sequence represented by SEC. ID. No. 2. Particularly, it is preferable to use a transformed transformant with an expression vector constructed by connecting the nucleotide sequence represented by SEC. ID. No. 4 continuously to a position just before the nucleotide sequence represented by SEC. ID. No. 1

Chondroitin polymerase can be collected from the material grown by protein extraction and purification procedures based on the form of the chondroitin polymerase produced.

For example, when chondroitin polymerase is produced in soluble form secreted in a medium (culture medium supernatant), the medium can be collected and used directly as chondroitin polymerase. In addition, when chondroitin polymerase is produced in soluble form secreted in the cytoplasm or in insoluble form (membrane fixation), chondroitin polymerase can be removed by cell disintegration such as a procedure using a nitrogen cavitation apparatus, homogenization , glass ball mill, ultrasonic treatment, an osmotic shock method, freeze-thaw, etc., surfactant extraction, a combination thereof or the like, and the extract can be used directly as chondroitin polymerase.

Chondroitin polymerase can be further purified in the medium or extracts, which is preferable. The purification may be imperfect purification (partial purification) or perfect purification, which may be appropriately selected based, for example, on the object using chondroitin polymerase, and the like.

Examples of the purification procedure include salting with ammonium sulfate, sodium sulfate, etc., centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, reverse phase chromatography, gel filtration, gel penetration chromatography , affinity chromatography, electrophoresis, a combination thereof and the like.

Chondroitin polymerase production can be confirmed by analyzing its amino acid sequence, actions, substrate specificity and the like.

<6> Synthesizing agent of the present invention

The synthesizing agent of the present invention is a sugar chain synthesizing agent, comprising the protein of the present invention and having chondroitin polymerase activity.

The synthesizing agent of the present invention is the result of applying the "GalNAc transfer action", "GlcNAc transfer action" and "GlcUA transfer action" of the enzyme of the present invention and the protein of the present invention as a sugar chain synthesizing agent.

The synthesizing agent of the present invention is used for the synthesis of sugar chains. The term "sugar chain synthesis", as used herein means a concept that includes the elongation of a certain sugar chain by transferring and adding a monosaccharide to said chain. For example, a concept of transfer and addition of a monosaccharide such as GlcUA, GalNAc, GlcNAc or similar to a sugar chain such as chondroitin, chondroitin sulfate, hyaluronic acid and the like to lengthen the sugar chain is included in the expression " sugar chain synthesis "as used herein.

The form of the synthesizing agent of the present invention is not limited, and may be any of a solution form, a frozen form, a lyophilized form and an immobilized enzyme form in which it is attached to a vehicle. In addition, it may contain other compounds (for example, pharmaceutically acceptable carrier, vehicle that is acceptable for the reagent, etc.), as long as it has no influence on the activity of chondroitin polymerase.

<7> Method of producing the sugar chain of the present invention

The sugar chain production process of the present invention utilizes the synthesizing agent of the present invention and is divided into the following five methods in response to sugar donor and acceptor substrates.

(1) Production process of the sugar chain of the present invention 1

Process for producing a sugar chain represented by the following formula (3), which comprises at least one step that allows the synthesizing agent of the present invention to contact a GalNAc donor and a sugar chain represented by the formula ( 1) following:

GlcUA-X-R1 (1) GalNAc-GlcUA-X-R1 (3)

(2) Production method of the sugar chain of the present invention 2

Method for producing a sugar chain represented by the following formula (4), which comprises at least one step that allows the synthesizing agent of the present invention to contact a GlcNAc donor and a sugar chain represented by the formula ( 1) following:

GlcUA-X-R1 (1) GlcNAc-GlcUA-X-R1 (4)

(3) Production process of the sugar chain of the present invention 3

Process for producing a sugar chain represented by the following formula (5), which comprises at least one step that allows the synthesizing agent of the present invention to contact a GlcUA donor and a sugar chain represented by the formula ( 2) following:

GalNAc-GlcUA-R2 (2) GlcUA-GalNAc-GlcUA-R2 (5)

(4) Method of producing the sugar chain of the present invention 4

Process for producing a sugar chain represented by the following formula (6) and (8), comprising at least one step that allows the synthesizing agent of the present invention to contact a GalNAc donor and a GlcUA donor and a sugar chain represented by the following formula (1):

GlcUA-X-R1 (1) (GlcUA-GalNAc) n-GlcUA-X-R1 (6) GalNAc- (GlcUA-GalNAc) n-GlcUA-X-R1 (8)

(5) Production process of the sugar chain of the present invention 5

Method for producing a sugar chain represented by the following formulas (7) and (9), comprising at least one step that allows the synthesizing agent of the present invention to contact a GalNAc donor, a GlcUA donor and a sugar chain represented by the following formula (2):

GalNAc-GlcUA-R2 (2) (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (7) GlcUA- (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (9)

In each of the formulas, X represents GalNAc or GlcNAc, and R1 and R2 each represents any group. R1 and R2 are the same or different from each other.

Examples of R1 and R2 include a sugar chain having a chondroitin skeleton, a sugar chain having a hyaluronic acid skeleton and the like.

The sugar chain represented by formula (1) is preferably chondroitin sulfate (specifically chondroitin sulfate C), chondroitin or hyaluronic acid having GlcUA in its non-reduced terminal, or an oligosaccharide thereof.

The sugar chain represented by formula (2) is preferably chondroitin sulfate (specifically chondroitin sulfate C) or chondroitin having GalNAc in its non-reduced terminal, or an oligosaccharide thereof.

As a GalNAc donor, a GalNAc nucleoside diphosphate is preferable, and in particular UDP-GalNAc is preferable. In addition, as a donor of GalNAc, a GlcNAc nucleoside diphosphate is preferable, and in particular UDP-GlcNAc is preferable. On the other hand, as a donor of GlcUA, the nucleoside diphosphate of GlcUA is preferable, and in particular UDP-GlcUA is preferable.

The process for making "contact" is not particularly limited, provided that the enzymatic reaction is generated by mutual contact of the enzyme molecules of the present invention (or the protein of the present invention) contained in the synthesizing agent of the present. invention, a donor and an acceptor (sugar chain). For example, the contact can be made in a solution in which the three components dissolve. In addition, the enzymatic reaction can be carried out continuously using an immobilized enzyme in which the chondroitin polymerase contained in the synthesizing agent of the present invention is linked to an appropriate solid phase (beads or the like) or to a membrane-type reactor using membrane ultrafiltration, dialysis membrane or the like. In addition, the enzymatic reaction can be performed by connecting the acceptor to a solid phase similar to the method described in WO 00/27437. In addition, a bi-reactor that regenerates (synthesizes) the donor can be used in combination.

Furthermore, in the above procedures (4) and (5), it is not always necessary to contact the GalNac donor and the GlcUA donor simultaneously with the synthesizing agent of the present invention and the sugar chain represented by formulas (1) or (2), and donors can come into contact alternately.

The conditions for carrying out the enzymatic reaction are not specifically limited, as long as they are conditions under which the enzyme of the present invention (or the protein of the present invention) can function, but it is preferable to carry out the reaction at pH. approximately neutral (for example, approximately pH 7.0 to 7.5) and it is more preferable to carry out the reaction in a buffer with a buffering action under pH. In addition, the temperature in this case is not specifically limited, as long as the activity of the enzyme of the present invention (or the protein of the present invention) is maintained, but a temperature of about 25 ° C to 30 ° C can serve as an example. In addition, when there is a substance that increases the activity of the enzyme of the present invention (or the protein of the present invention), the substance can be added. For example, it is preferable to let Mn2 + and the like coexist. The duration of the reaction may optionally be determined by those skilled in the art in response to the amounts of the synthesizing agent of the present invention, the donor and the acceptor to be used and other reaction conditions.

The isolation and the like of the sugar chain from the product formed can be carried out by known procedures.

In addition, a sulfated saccharide such as chondroitin sulfate or the like can be produced using the synthesizing agent of the present invention (chondroitin polymerase) and a sulfate transferase in combination.

For example, a sulfated saccharide such as chondroitin sulfate or the like can be produced simultaneously carrying out the formation of chondroitin and the transfer of the sulfate group in the production process of the above sugar chain, also allowing a donor of the sulfate group to coexist (3'-phosphoadenosine 5'-phosphosulfate (PAPS) or the like) and a sulfotransferase. The sulfotransferase can be used as an immobilized enzyme by attaching it to an appropriate solid phase (beads or similar) similar to the previous case or it is allowed to react continuously using a membrane-type reactor that uses ultrafiltration membrane, dialysis membrane

or similar. In this case, a bioreactor that regenerates (synthesizes) the sulfate group donor can be used in combination.

The sulfotransferase that can be used herein can be any enzyme that can transfer a sulfate group to chondroitin and can be appropriately selected from known enzymes based on the type of chondroitin sulfate desired. In addition, two or more types of sulfotransferases that have different transfer positions of the sulfate group can be used in combination.

Chondroitin 6-O-sulfotransferase (J. Biol. Chem., 215 (28), 21075-21080 (2000)) can serve as an example as sulfotransferase. However, there is no limitation to this and other enzymes can also be used.

<8> Probe of the present invention

The probe of the present invention is a hybridization probe comprising a nucleotide sequence complementary to the nucleotide sequence represented by the SEC. ID. No. 1

The probe of the present invention can be obtained by preparing an oligonucleotide comprising a nucleotide sequence complementary to the nucleotide sequence represented by the SEC. ID. No. 1, and marking it with a suitable marker for hybridization (for example, radioisotope).

The size of the oligonucleotide is appropriately selected based on the conditions and the like of the hybridization used by the probe of the present invention.

It is expected that the probe of the present invention will become a useful tool for examining the biological functions of chondroitin sulfate. This is because chondroitin sulfate is widely expressed and plays an important role in a large number of tissues, particularly in the brain. It is considered that the probe also serves to search for the relationship between genes and diseases.

<9> Catalyst of the present invention

The catalyst of the present invention is a glycosyl transfer catalyst comprising the protein of the present invention and having a chondroitin polymerase activity.

The catalyst of the present invention is a result of applying the "transfer action of GalNAc", "the transfer action of GlcNAc", and "transfer action of GlcUA" of the enzyme of the present invention and of the protein of the present invention as a glycosyl transfer catalyst.

The catalyst of the present invention can be used for the transfer of GlcUA, GalNAc or GlcNAc. For example, it can be used to transfer a monosaccharide, such as GlcUA, GalNAc or GlcNAc or similar to a non-reduced terminal of a sugar chain such as chondroitin, chondroitin sulfate, hyaluronic acid and the like.

The catalyst form of the present invention is not restricted, and can be any of a solution form, a frozen form, a lyophilized form and an immobilized enzyme form in which it is attached to a vehicle. In addition, it may contain other components (eg, pharmaceutically acceptable carrier, vehicle that is acceptable for reagent, etc.), as long as it has no influence on the transfer activity of GlcUA, GalNAc or GlcNAc.

Since the enzyme of the present invention and the protein of the present invention can transfer GlcUA and GalNAc alternately, as a single molecule, they are remarkably useful as tools for large-scale production of the sugar chain having a chondroitin skeleton ( chondroitin, chondroitin sulfate, and the like), as active ingredients of the synthesizing agent of the present invention and the catalyst of the present invention, reagents or the like. In addition, the DNA of the present invention is very useful as a tool for large-scale production of said enzyme of the present invention and of the protein of the present invention. The vector of the present invention is very useful, because it can hold the DNA of the present invention stably and function effectively and efficiently. The transformant of the present invention is also very useful, because not only can it retain the vector of the present invention in a stable manner and function effectively and efficiently, but it can also be used as such for the large-scale production of the enzyme of the present invention. invention and protein of the present invention. In addition, the enzyme production process of the present invention is very useful for large-scale production of the enzyme of the present invention and of the protein of the present invention. Also, the sugar chain production process of the present invention is very useful for large-scale production of the sugar chain having the chondroitin skeleton (chondroitin, chondroitin sulfate, and the like). The synthesizing agent of the present invention and the catalyst of the present invention are very useful, because they can be used in the sugar chain production process of the present invention.

Since high quality and uniform chondroitin polymerase can be produced conveniently and on a large scale by the present invention, low cost products can be provided for the industrial field and therefore the present invention has very high availability.

The present invention is explained in detail based on the examples, however, the present invention is not limited thereto.

UDP- [14C] GlcUA, UDP- [3H] GalNAc and UDP- [14C] GlcNAc used in the examples were purchased from NEN Life Sciences. In addition, UDP-GlcUA, UDP-GalNAc and UDP-GlcNAc were purchased from Sigma.

Example 1

Chondroitin polymerase gene cloning:

(1) Preparation of the DNA bank

The K4 strain (serotype O5: K4 (L): H4, ATCC 23502) of Escherichia coli, was grown at 37 ° C overnight in 50 ml of LB medium. The cells were collected by centrifugation (3,800 rpm, 15 minutes), suspended in 9 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) (hereinafter referred to as " TE ") and then treated at 37 ° C for 1 hour, adding 0.5 ml of 10% SDS and 50 µl of proteinase K (20 mg / ml, Boehringer Mannheim). To the suspension, 10 ml of PCI solution (phenol: chloroform: isoamyl alcohol = 25: 24: 1) was added, followed by stirring for 30 minutes, and the resulting mixture was centrifuged (3,800 rpm, 15 minutes) to collect the layer of water and insoluble matter from the intermediate layer and centrifuged (10,000 rpm, 30 minutes) again. The supernatant was recovered and 50 µl of RNase A (20 mg / ml, Sigma) was added thereto for the reaction at 37 ° C for 1 hour. To the treated solution was added 10 ml of PCI solution, followed by stirring for 30 minutes, and the resulting mixture was centrifuged (3,800 rpm, 15 minutes), to collect the water layer and centrifuged (10,000 rpm, 30 minutes) another time. The supernatant was recovered and dialyzed against 2,000 ml of TE at 4 ° C overnight, and the dialyzed solution (7.5 ml) was used as a chromosomal DNA solution (DNA concentration, 0.9 mg / ml ). The chromosomal DNA solution (120 µl) from strain K4 obtained in this way was digested using a Sau3A1 restriction enzyme (4 units: NEB) at 37 ° C for 30 minutes and then subjected to 0-agarose gel electrophoresis, 3%, and then the agarose gel corresponding to the DNA of approximately 7 to 11 kbp was cut. The gel cut in this way was placed in a 1.5 ml capacity tube with a hole in its bottom chopped with a needle and, together with the tube, was inserted into a 2 ml capacity tube and centrifuged (8,000 rpm, 1 minute) to break the gel. The neutralized phenol in almost the same gel volume was added thereto, followed by intense stirring and then the resulting mixture was frozen at -80 ° C. Thirty minutes later, the temperature was returned to room temperature and the mixture was melted, followed by centrifugation (13,000 rpm, 5 minutes). The resulting aqueous layer was collected, the same volume of PCI solution was added thereto followed by stirring, and then the mixture was centrifuged

(13,000 rpm, 5 minutes). The aqueous layer was collected, 1/10 volume of 3M sodium acetate solution and the same volume of 2-propanol was added thereto to precipitate the DNA, and the precipitate was then collected by centrifugation (13,000 rpm, 30 minutes ). To the precipitate collected in this way, 70% ethanol solution was added, followed by centrifugation (13,000 rpm, 5 minutes), and then the resulting precipitate was dissolved by adding 100 µl of TE. To concentrate the resulting solution, the DNA was precipitated by adding 10 µl of 3M sodium acetate solution and 300 µl of ethanol and then recovered by centrifugation (13,000 rpm, 20 minutes). To the precipitate collected in this way, 70% ethanol solution was added, followed by centrifugation (13,000 rpm, 5 minutes), and the resulting precipitate was dissolved in 4 µl of purified water to obtain a chromosomal DNA fragment solution. The DNA fragment solution (2 L) was inserted into a phage vector λ (λ EMBL3: STRATAGENE) that had been treated with restriction enzymes (BamHi (80 units: NEB) and EcoRI (80 units: NEB)) and It was subjected to packaging using a packing kit (Gigapack III Gold Packaging Extract, STRATAGENE) according to the manufacturer's instructions, and then the Escherichia coli strain (NM 539) was infected with phage λ and propagated to prepare the DNA bank K4 chromosome.

(2) Probe preparation

Between 3 regions of the rest of the K antigen gene group of Escherichia coli strain K5 (serotype 010: K5 (L): H4, ATCC 23506) having known sequences (Mol. Microbiol., 17 (4), 611-620 (1995)), while interposing the specific R-II region of the K antigen polysaccharide (gene bank registration number X77617), a primer series (CS-S 5'-ACCCAACACTGCTACAACCTATATCGG-3 '(SEC. ID No. 5); CS-AS 5'GCGTCTTCACCAATAAATACCCACGAAACT-3 '(SEQ ID No. 6)) to obtain a DNA fragment of approximately 1 kbp from the 3' terminal of the RI region (gene bank registration number X74567 ) and another primer series (TM-S 5'-CGAGAAATACGAACACGCTTTGGTAA-3 '(SEQ. ID. No. 7); TM-AS 5'-ACTCAATTTTCTTTCAGCTCTTCTTG-3' (SEQ. ID. No. 8)) to obtain the fragment of DNA of approximately 1 kbp from the 5 'terminal of the R-III region (gene bank registration number X53819) was selected and prepared.

Using the respective primer sets for immune response for RI and R-III and using, as a template, genomic DNA fragments of strain K4 extracted and purified after treatment with Sau3AI and subsequent agarose gel electrophoresis in section (1) above, the polymerase chain reaction (PCR) was carried out (94 ° C, 1 min. - (94 ° C, 45 s-47 ° C, 30 s-72 ° C, 5 min.) 30 cycles-72 ° C, 10 min. (for RI), 94 ° C, 1 min .- (94 ° C, 45 s-50 ° C, 30 s-72 ° C, 5 min.) 30 cycles-72 ° C, 10 min. (For R-III) to obtain the DNA fragment of the RI region of 1.3 kbp (K4RI3) from K4 of the R-III region of 1.0 kbp (K4RIII5) Nucleotide sequences of the DNA fragments obtained in this way were determined using the ABI PRISM 310 genetic analyzer (Perkin- Elmer.) Homology with the K5 strain of DNA at the same genetic positions was 96% and 95%, respectively.

(3) Genetic cloning of the K4R-II region

Using the DNA fragments (K4RI3 and K4RIII5) of the respective R-I region and R-III region as probes, the gene groups of the K4 antigen of the K4 chromosomal DNA bank obtained in section (1) above were identified. The culture of Escherichia coli (strain NM539) (30 µl) was infected with the K4 chromosomal DNA bank (40 µl of phage λ) (37 ° C, 15 minutes), 10 ml of agarose from the top were added thereto, the The resulting mixture was spread on the medium in the LB plate contained in five 10 x 14 cm Petri dishes, followed by cultivation at 37 ° C for 9.5 hours to form plates. Two 9 x 13 cm membranes (Hybond-N +: Amersham Pharmacia Biotech) were prepared for each plate, and the first and second membranes were placed in the middle for 1 minute and 3 minutes, respectively. After removing excess moisture, each membrane was soaked for 2 minutes in 0.5 M NaOH containing 1.5 M NaCl to carry out the denaturation treatment and then soaked for 3 minutes in 1 M Tris-HCl ( pH 7.4) containing 1.5 M NaCl to carry out the neutralization treatment. After drying, the membrane was dried at 80 ° C for 2 hours to prepare a filter. The filter was subjected to prehybridization treatment at 65 ° C for 1 hour, hybridized with [32P] K4RI3 at 64 ° C overnight (15 hours) in 0.5 M Church phosphate buffer (pH 7.2), 1 mM EDTA and 7% SDS, and then washed three times with 40 mM Church phosphate buffer (7.2) containing 1% SDS (65 ° C, each for 15 minutes). The filter was dried and then exposed to an X-ray film to thereby obtain 30 positive plates. The presence of K4RI3 was confirmed for each of them by PCR, and 7 plates between them underwent a second identification. Next, the filter hybridized with K4RI3 was boiled in 0.5% SDS solution for 3 minutes to remove K4RI3 and then dried to be used as a K4RIII5 hybridization filter. The filter was subjected to prehybridization treatment at 65 ° C for 1 hour, hybridized with [32P] K4RIII5 at 64 ° C overnight and then washed three times with 40 mM Church phosphate buffer containing 1% SDS. The filter was dried and then exposed to an x-ray film to thereby obtain 29 positive plates. The presence of K4RIII5 was confirmed for each of them by PCR, and 18 plates among them underwent a second identification. In the second identification, de 9 cm LB plate medium was used, and positive plates were obtained by the same procedure of the first identification.

After the first and second identification, 4 λ phage clones of the R-I region and 10 clones of the R-III region were obtained. Each of the clones was subjected to enzymatic treatment with EcoRI (10 units: NEB), SaII (10 units: NEB) and BamHI (10 units: NEB) each independently or simultaneously in various combinations, and their restriction maps were prepared based on the size of the fragments observed by electrophoresis (figure 1).

Among these clones, a clone (CS23, 15.4 kbp insertion region) is a DNA clone prepared based on the R-III region probe, but since it also had a labile reaction with the RI region probe , the 5 'terminal sequence of the insertion region was examined to find a sequence that completely coincided with the 3' end of the probe with the RI region. Since both DNA fragments of the RI region and of the R-III region were contained in the insertion region, the clone was considered as a clone containing the entire R-II region of the K antigen genetic group of strain K4 .

(4)

Genetic analysis of the R-II region of K4

Subcloning the previous SC23 clone was performed to carry out its sequencing. First, each of the approximately 3 kbp and 8 kbp fragments, obtained by treating the SC23 clone with EcoRI and the 2 kbp, 5 kbp and 7 kbp DNA fragments, obtained by treating it with SalI was ligated with a vector of cloning (pBluescript II KS (-)) and was integrated into the strain (XLI-Blue) of Escherichia coli to obtain a clone with different direction of insertion. Repeating the "treatment of the multicloning points of the vector with the ligation transformation of several restriction enzymes" in the clone, 22 plasmids having partial fragments of R-II region DNA were obtained. By sequencing the DNA insertion fragments and connecting them, the complete genetic sequence of the R-II region of K4 was determined (SEQ. ID. # 3).

(5)

Chondroitin polymerase gene identification

As a result of the analysis of the DNA sequence of the R-II region of strain K4, the presence of 8 open reading frames (ORF) was predicted (Figure 2).

Among them, the ORF in third position counting from the R-III side (2,061 bp (nucleotides numbers 3,787 to 5,847 in SEQ ID No. 3, the 2058 bp sequence excluding the termination codon is shown in SEQ. ID No. 1), 686 as the number of amino acids, molecular weight obtained by calculation 79,276 (SEQ. ID. No. 2)) presented 59% homology with a Pasteurella multocida hyaluronic acid synthase (pmHAS class 2 type; J. Biol. Chem., 273 (14), 8454-8458 (1998)). In addition, the ORF count in the first position from the R-III side (1,017 bp (nucleotides numbers 643 to 1,649 in SEQ ID No. 3, 339 as number of amino acids) had 60% homology with UDP- 4-epimerase of Pasteurella multocida (proposal (29-OCT.-1996) Genetics and Microbiology, Autonomous University of Barcelona, Building C, Bellaterra, BCN 08193, Spain), ORF in fourth position (1,332 bp (nucleotides numbers 5,877 to 7,207 in SEQ ID NO. 3)) had great homology (98%) with the insertion sequence 2 (Nucleic Acids Res., 6 (3), 1,111-1,122 (1979)) and the ORF in seventh position (1,167 bp ( Nucleotides numbers 11,453 to 12,619 in SEQ ID No. 3), 389 as the number of amino acids) had 65% homology with the kfiD gene (Mol. Microbiol., 174 (4), 611-620 (1995)) (encodes UDP-glucose-dehydrogenase) of the Escherichia coli strain (K5) In addition, given that the ORF in eighth position (1,035 bp (nucleotides numbers 13,054 to 14,088 in SEC. ID No. 3), 345 as the number of amino acids) containing a common DXD motif to glucosyltransferase, was considered to have sugar transfer activity. As for the remaining three ORFs (No. 2, No. 5 and No. 6 (nucleotides numbers 1,849 to 3,486, 7,210 to 8,673 and 9,066 to 10,631, respectively, in SEQ. ID. No. 3), no homology was found.

Example 2

Expression and enzymatic activity of chondroitin polymerase protein

(1) To confirm that the ORF of the R-II region of K4 (# 3) is a chondroitin polymerase gene, primers were prepared with restriction enzyme cut points and that they interpose the corresponding ORF residue ( K4CSP 5'-CGGGATCCCGATGAGTATTCTTAATCAAGC-3 '(SEQ. ID. No. 9); K4C-AS 5'GGAATTCCGGCCAGTCTACATGTTTCC-3' (SEQ. ID. No. 10)) and the PCR (94 ° C, 1 min.) ( 94 ° C, 30 s-59 ° C, 30 s-74 ° C, 3 min.) 20 cycles-74 ° C, 10 min.). The PCR product was subjected to 0.7% agarose gel electrophoresis and extracted and purified using a gel extraction kit (QIAGEN). After treatment with the restriction enzymes (BamHI and EcoRI), the product was again subjected to 0.7% agarose gel electrophoresis and extracted and purified in the same way to be used as an insert.

The insert prepared in the previous paragraph was inserted into an expression vector (pTrcHisC: Invitrogen; containing the nucleotide sequence represented by SEQ. ID. No. 4) that had been treated with the restriction enzymes (BamHI and EcoRI) and CIP, at 16 ° C for 1 hour in the presence of T4 DNA ligase, and it was transformed into Escherichia coli strain (TOP10). By culturing the Escherichia coli strain (medium of the LB plate containing ampicillin, 37 ° C, overnight), 7 colonies were obtained. A clone containing a plasmid in which the previous insert was inserted correctly was selected from among them. Escherichia coli (at 37 ° C overnight) was grown in 1.5 ml of LB medium containing amplicillin (100 µg / ml) and 50 µl of the suspension of the cultured cells was inoculated in 50 µl of LB medium containing amplicillin ( 100 µg / ml) and was grown at 37 ° C until the OD600 was 0.6. To the culture was added 1 ml of 0.5 M isopropyl 1-thio-β-D-galactoside (IPTG) (final concentration: 1 mM) and the induction was performed at 37 ° C for 3 hours. Cells were collected by centrifugation (10,000 rpm, 30 minutes) and suspended by adding 4 ml of a lysis buffer (50 mM NaB2PO4 (pH 8.0) containing 300 mM NaCl and 10 mM imidazole). To the suspension, 4 mg of lysozyme (Sigma) was added, the resulting mixture was allowed to stand on ice for 30 minutes and then the cells were destroyed three times by ultrasonic exposure, each for 10 seconds, using an ultrasonic bath . The supernatant was collected by centrifugation

(10,000 rpm, 30 minutes) and applied to the agarose column with Ni-NTA (1 ml of vehicle, equilibrated with lysis buffer, QIAGEN), followed by stirring with a C agitator for 1 hour. The vehicle was submerged by installing the column and then the column was washed twice using 4 ml for each, one was a buffer (50 mM NaH2PO4 (pH 8.0) containing 300 mM NaCl and 20 mM imidazole). The proteins were then eluted by passing 4 times 0.5 ml of each of an elution buffer (50 mM NaH2PO4 (pH 8.0) containing 300 mM NaCl and 250 mM imidazole). The eluate containing the protein of interest (1 ml) was dialyzed at 4 ° C for 2 days against 500 ml of PBS (phosphate buffered saline) containing 20% glycerol to thereby obtain approximately 0.5 ml (content 0.4 mg / ml protein) of a dialyzed solution (enzyme solution of the present invention (protein of the present invention)).

Western immunoblotting of the protein obtained in this way was carried out by electrophoresis in sodium dodecyl sulfate with polyacrylamide gel (SDS-PAGE). In the SDS-PAGE 10% gel was used. The protein was detected by staining with Coomassie bright blue. Western blotting was performed by transferring the protein to the SDS-PAGE gel on a nitrocellulose membrane, blocking the membrane with 5% skim milk (dissolved in 25 mM Tris-HCl (pH 7.5) containing 150 mM NaCl and Tween 20 0.05% (this solution was called TBS-T) and then treated with anti-tetra-His antibody (Qiagen) After washing several times with TBS-T, this membrane was treated with peroxidase-bound anti-mouse IgG After washing with TBS-T, the reacted protein was detected by the ECL detection system (Amersham).

As a result, the protein had a band at about 80 kDa by Western blot analysis using SDS-PAGE and anti-tetra-His antibody. On the other hand, an immunologically reactive band (expression vector without insertion) was not detected in the culture extract of a reference.

(2) Enzymatic activity analysis (GalNAc transfer activity analysis)

The enzyme of the present invention (2 µg) shark cartilage chondroitin sulfate C hexasaccharide, purified by degradation with testicular hyaluronidase, as acceptor (70 pmol) and UDP-GalNAc (3 nmoles), UDP-GlcUA (3 nmoles) and UDP- [3H] GalNAc (0.1 nmol, 0.1 µCi) were added as donors to 50 mM Tris-HCl (pH 7.2) containing 20 mM MnCl2, (0.1 M NH4) 2SO4 and ethylene glycol 1 M and the total volume was adjusted to 50 µl, and then the reaction was carried out at 30 ° C for 30 minutes and the enzyme was thermally inactivated. To the reaction solution, 3 volumes of 95% ethanol containing 1.3% potassium acetate were added, and the sample was centrifuged at 10,000 x g for 20 minutes. The precipitate was dissolved in 50 µl of distilled water and applied to a Superdex peptide column (300 x 10 mm: Amersham Biosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml / min) and The eluate was fractionated to 0.5 ml and the radioactivity ([3 H] count) of each fraction was measured using a scintillation counter. The chondroitin synthesizing activity was determined by calculating the amount of radioactivity incorporated in the molecular weight fractions higher than the acceptor substrate. The results are shown in Figure 3. In Figure 3, the black squares indicate radioactivity when chondroitin sulfate C hexasaccharide was used as the acceptor, blank triangles indicate radioactivity when the product of the enzymatic reaction was treated with ABC chondroitinase and black circles indicate the reference (in which the thermally inactivated enzyme of the present invention was used).

As a result, the elution position of the radioactivity appeared on the side of the molecular weight higher than that of the chondroitin sulfate hexasaccharide C (the wide peak that has its upper part at around 5,000 Da) (black squares in the figure 3). In addition, when the product of the enzymatic reaction was treated with chondroitinase ABC, the peak of the high molecular weight shifted to a position corresponding to the disaccharide fraction (blank triangles in Figure 3). When the disaccharide composition of this ABC chondroitinase digest was analyzed using a high performance liquid chromatography (HPLC), only one unsaturated and unsulfated chondroitin disaccharide (ΔdiOS) was detected.

In addition, the product of the enzymatic reaction was digested completely by treatment with ACII chondroitinase as well, but was not digested by Streptomyces hyaluronidase or by heparitinase I.

Based on the foregoing, it was shown that the enzyme of the present invention thus obtained at least transfers GalNAc to the hexasaccharide of chondroitin C sulfate from UDP-GalNAc (donor). This specific activity was 3.25 ± 0.64 nmoles of GalNAc / min / mg protein.

(3) Analysis of the product size of the enzymatic reaction

The product size of the enzymatic reaction was examined by carrying out the enzymatic reaction and chromatography in the same manner as in "section (2) Analysis of enzyme activity" above. The results are presented in Figure 4.

It was confirmed from Figure 4 that the enzyme of the present invention obtained above at least transfers GalNAc to an acceptor (oligosaccharide having a chondroitin skeleton (chondroitin C sulfate hexasaccharide prepared using testicular hyaluronidase)) from the GalNAc donor ( UDP-GalNAc) to thereby form chondroitin having a molecular weight of 10,000 to 20,000 or more.

(4) Donor substrate specificity analysis

Using UDP- [14C] GlcUA, UDP- [10C] GlcNAc or UDP- [3H] GalNAc as a donor, the transfer activity of the following acceptors was examined according to the procedure described in "section (2) Measurement of

5

10

fifteen

twenty

25

30

35

40

Four. Five

50 UDP- [3H] GalNAC UDP [14C] GlcNAc UDP [14C] GlcUA

UDP- [3H] GalNAc UDP [14C] GlcUA UDP [14C] GlcNAC UDP [14C] GlcUA

enzymatic activity "above. The products after the enzymatic reaction were analyzed by gel filtration.

The results of the use of the hexasaccharide or chondroitin sulfate heptasaccharide C as acceptor are shown in Figure 5. In addition, the black circle in Figure 5 indicates a reference (a case in which the thermally inactivated enzyme of the present invention).

(A) Chondroitin C sulfate hexasaccharide (purified product by degrading chondroitin C sulfate from shark cartilage with testicular hyaluronidase, and the non-reduced terminal is GlcUA).

Heptasaccharide was synthesized only when UDP-GalNAc was used only as a donor (black pills in Figure 5).

Substantial transfer was not found when UDP-GlcUA was used only as a donor (black triangle in Figure 5).

Although very little transfer of GlcNAc was found when UDP-GlcNAc was used only as a donor. This transfer activity was 6.3% based on 100% activity in the case of the use of UDP-GalNAc as a donor (square in black in Figure 5). However, polysaccharides having an octasaccharide size or more were not obtained even when both UDP-GlcNAc and UDP-GlcUA were used together with it.

In addition, since the incorporation of radioactivity was not in the absence of the acceptor substrate, it was suggested that the acceptor substrate is essential for the synthesis of chondroitin by this enzyme.

(B) Chondroitin C sulfate heptasaccharide (product obtained by allowing the enzyme of the present invention to react with the above chondroitin C sulfate hexasaccharide and thereby joining a GalNAc moiety to the non-reduced terminal of the hexasaccharide).

Octasaccharide was synthesized only when UDP-GlcNAc was used as a donor (blank triangle in Figure 5).

When UDP-GalNAc or UDP-GlcNAc was used only as a donor, no substantial transfer was found in each case (respectively, blank and blank square pill in Figure 5).

In addition, the results of carrying out similar tests independently each are shown in Table 1. In addition, the term "SC" in Table 1 means chondroitin C sulfate. In addition, the parentheses in the Table show the length of the sugar chain after the enzyme reaction and "-" means that sugar labeled with UDP It was not transferred to the corresponding acceptor sulfate.

Table 1

Specific chondroitin polymerase activity (nmoles / min / mg protein)

Substrate of the donor Substrate of the acceptor

Sugar labeled with UDP Sugar unlabeled with UDP Hexasaccharide of SC Heptasaccharide of SC

none none none

UDP-GlcUA UDP-GalNAc UDP-GlcUA UDP-GlcNAc

From the above results, it was shown that the enzyme of the present invention obtained above transfers GalNAc from UDP-GlcNAc to a sugar chain (acceptor) having a chondroitin skeleton whose non-reduced terminal is GlcUA. In addition, it was shown that when the acceptor is used, the enzyme of the present invention obtained in the foregoing exhibits the activity to transfer GlcNAc from UDP-GlcNAc, but the activity is much less than its GalNAc transfer activity.In addition, it is demonstrated that when the acceptor is used, the enzyme of the present invention obtained in the foregoing has substantially no activity to transfer GlcUA from UDP-GlcUA. Based on these results, it was shown that the previous enzyme of the present invention is not capable of transferring GlcUA to the non-reduced GlcUA terminal but is capable of transferring a remaining GalNAc (or, quite lightly, GlcNAc).

Furthermore, it was shown that the enzyme of the present invention obtained in the foregoing transfers GlcUA from UDP-GlcUA to a sugar chain (acceptor) having a chondroitin skeleton whose non-reduced terminal is GalNAc. In addition, it was shown that when the acceptor is used, the enzyme of the present invention obtained above has substantially no activities to transfer GalNAc from UDP-GalNAc and to transfer GlcNAc

0.59 ± 0.16 (hepta) 0.04 ± 0.02 (hepta) 0.0 ± 0.0 (-) 3.25 ± 0.64 (poly) 2.75 ± 0.28 (poly) 0.05 ± 0.02 (poly) 0.0 ± 0.0 (-)

0.0 ± 0.0 (-) 0.0 ± 0.0 (-) 0.53 ± 0.08 (octa) not measured not measured not measured not measured

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from UDP-GlcNAc. Based on these results, it is demonstrated that the previous enzyme of the present invention is not capable of transferring GalNAc to the unreduced GalNAc terminal but is capable of transferring a GlcUA moiety.

Based on the foregoing, it was shown that the above enzyme of the present invention is capable of transferring GlcUA and GalNAc alternately, from a GlcUA donor and a GalNAc donor, respectively, to the non-reduced terminal of the sugar chain.

(5) Specificity analysis of the acceptor substrate

Using tetrasaccharide (140 pmoles) or hexasaccharide (140 pmoles) of degraded and purified chondroitin C sulfate using testicular hyaluronidase, tetrasaccharide (260 pmoles) or hexasaccharide (175 pmoles) of chondroitin degraded and purified by the method of Nagasawa (Carbohydrate Research, 97, 263-278 (1981)), hexasaccharide (175 pmoles) of degraded and purified hyaluronic acid using testicular hyaluronidase, chondroitin C sulfate (molecular weight 20,000), chondroitin (molecular weight 10,000), dermatan sulfate (molecular weight 15,000) , hyaluronic acid (molecular weight 20,000), or heparin (molecular weight 10,000) as acceptor, transfer activity was examined by the following method. In addition, sugar chains were purchased from Seikagaku Corporation.

The enzyme of the present invention (2 µg), UDP-GalNAc (Sigma) (60 pmoles), UDP-GlcUA (Sigma) (0.6 nmoles) and UDP- [3H] GalNAc (0.1 nmoles, 0.1 µCi) as donors of each of the above sugar chains as acceptor were added to 50 mM Tris-HCl containing 20 mM MnCl2, 0.1 M (NH4) 2SO4 and 1 M ethylene glycol, and the total volume was adjusted to 50 µl, and then the enzymatic reaction was carried out at 30 ° C for 30 minutes and the enzyme was thermally inactivated. The reaction solution was applied to a Superdex Peptide column (300 x 10 mm: Amersham Biosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml / min) and the eluate was fractionated at 0, 5 ml and the radioactivity ([3 H] count) of each fraction was measured using a scintillation counter. The results are presented in Table 2. The numbers in brackets in the Table are relative values when the amount of radioactivity (amount of GalNAc transferred) by using the hexasaccharide of chondroitin C sulfate as acceptor was defined as 100%.

Table 2

Acceptor substrate Specific incorporation activity of [3 H] nmoles / min / mg protein%

Chondroitin C Sulfate

Tetrasaccharide 1.44 ± 0.24 43.0

Hexasaccharide

3.34 ± 0.50 100.0

Polysaccharide (PM 20,000)

3.41 ± 0.48 100.0

Chondroitin

Tetrasaccharide 1.12 ± 0.21 33.5

Hexasaccharide

1.24 ± 0.45 37.0

Polysaccharide (PM 10,000)

0.53 ± 0.13 15.8

Hyaluronic acid

Hexasaccharide 0.80 ± 0.15 24.0

Polysaccharide

0.27 ± 0.02 8.2

(PM 20,000)

Dermatan sulfate

Polysaccharide 0.06 ± 0.02 1.9

(PM 15,000)

Heparin

Polysaccharide 0.0 ± 0.0 0.0

(PM 10,000)

Based on the above results, it was shown that chondroitin C sulfate hexasaccharide becomes the most suitable acceptor substrate. Chondroitin hexasaccharide also functioned as an acceptor substrate, but its activity was low (37%) compared to the case of chondroitin sulfate hexasaccharide C. The incorporation into chondroitin sulfate tetrasaccharide or chondroitin tetrasaccharide was the same (43% and 33.5%, respectively). To the surprise of the authors, hyaluronic acid hexasaccharide and hyaluronic acid (molecular weight 20,000) also functioned as acceptor substrates. The level of incorporation of chondroitin C sulfate (molecular weight 20,000) was similar to that of chondroitin C hexasaccharide. The incorporation was not so high in the case of chondroitin (molecular weight 10,000).

Summarizing the above results, it was shown that the above enzyme of the present invention uses oligosaccharides and polysaccharides having a chondroitin skeleton (at least tetrasaccharide, hexasaccharide and heptasaccharide of chondroitin C sulfate, tetrasaccharide and hexasaccharide chondroitin, chondroitin C sulfate ( molecular weight 20,000) and chondroitin (molecular weight 10,000)) and oligosaccharides and polysaccharides of hyaluronic acid (at least hexasaccharide of hyaluronic acid and hyaluronic acid (molecular weight 20,000)) as acceptors.

On the other hand, the incorporation of radioactivity was not found in dermatan sulfate (molecular weight 15,000) or heparin (molecular weight 10,000) which demonstrates that they do not function substantially as substrates

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55

acceptors

(6) Analysis of the influence of temperature

The enzymatic reaction was carried out in the same manner as in the "section (2) Analysis of enzyme activity" above, changing the temperature of the enzymatic reaction to 25, 30, 37, 40, 45 or 100 ° C, and the solution after The reaction was applied to a Superdex 74 column (30 x mm10 mm: Amersham Biosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml / min). The eluate was fractionated into 1 ml portions and the radioactivity ([3 H] count) of each fraction was measured using a scintillation counter. The results are shown in Figure 6. In addition, the pill, square, triangle, x and * marks in Figure 6 show the results of 25, 30, 37, 40, 45 or 100 ° C, respectively. In addition, the circle in Figure 6 presents the result of a reference (in which the thermally inactivated enzyme of the present invention was used).

As shown in Figure 6, under the reaction conditions and in the temperature range examined this time, the molecular weight of the reaction product became small as the temperature was increased. The highest incorporation was found at 30 ° C, but the product with the highest molecular weight was obtained at 25 ° C.

It is considered from the results that the enzymatic activity of the previous enzyme of the present invention decreases as the reaction temperature increases starting at 25 ° C under the reaction conditions of this time.

(7) Analysis of the influence of metal ions and the like

The enzymatic reaction was carried out in the same manner as in the "section (2) Analysis of enzyme activity" above, except that each of the various metal salts (MnCl2, FeCl2, MgCl2, CaCl2 or CuCl2) or EDTA was added instead of MnCl2, and the solution after the reaction was applied to a Superdex 75 column (300 x Φ100 mm. Amersham Biosciences, chromatography conditions; buffer: 0.2 M NaCl, flow rate: 0.5 ml / min). The eluate was fractionated in 1 ml and the radioactivity ([3 H] count) of each fraction was measured using a scintillation counter. Relative radioactivity values when MnCl2 is present defined as 100% are presented below.

Table 3

Metal salt Relative value of radioactivity (%) MnCl2 100.0 FeCl2 30.6 MgCl2 30.7 CaCl2 0.0 CuCl2 0.0 EDTA 0.0

Based on these results, the previous enzyme of the present invention had the highest activity in the presence of Mn2 + ion. In addition, in the presence of the Fe2 + or Mg2 + ion, it had approximately 30% activity compared to the case of the presence of the Mn2 + ion. In addition, it was shown that it does not act substantially in the presence of the Ca2 + or Cu2 + ion or ethylenediaminetetraacetic acid.

Furthermore, it was shown that the previous enzyme of the present invention acts in the presence of the Fe2 + or Mg2 + ion as well, and its enzymatic activity in the presence of the Mn2 + ion is greater than the enzymatic activity in the presence of the Fe2 + or Mg2 + ion.

(8)

optimal reaction pH

When the optimum reaction pH of the enzyme of the present invention was examined by changing the pH of the "section

(2)

Enzyme activity analysis "before several levels, was pH 7.0 to 7.5.

(9)

Relationship with enzymatic reaction time

By adjusting the enzymatic reaction time in 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours or 18 hours, the enzymatic reaction was performed in the same manner as in the "section (2) Analysis of enzyme activity" above, and Radioactivity incorporated into the product of the enzymatic reaction was analyzed. Gel filtration patterns of [3 H] GalNAc after several reaction periods are presented in Figure 7, and the total amounts of radioactivity incorporation after several periods of enzymatic reaction in Figure 8. The white circle, the circle black, the white triangle, the black triangle, the white square and the black square, present the results after 10 minutes, 30 minutes, 1 hour, 3 hours, 6 hours and 18 hours, respectively. In addition, the arrow with "20 k", "10 k", "5 k", "14", "8" or "6" presents the elution position of the molecular weight 20,000, 10,000, 5,000, tetradecasaccharide (molecular weight: approximately 2,800), octasaccharide (molecular weight: 1,600) or hexasaccharide (molecular weight: approximately 1,200) of hyaluronic acid (standard), respectively.

It was demonstrated from the results of Figure 8 that under the test conditions, the rapid incorporation of [3 H] GalNAc is found after 3 hours and 6 hours, but the incorporation becomes slow as the reaction approaches at 20 hours

Furthermore, from the results of Figure 7, the incorporation was shown to increase and a high molecular weight reaction product is obtained after a long period of reaction time.

In addition, a high molecular weight product was obtained quickly when the acceptor substrate (hexasaccharide) was placed at a lower concentration, and a low molecular weight product was obtained when the acceptor substrate (hexasaccharide) was placed at high concentration.

(10) Determination of the Michaelis constant (Km)

The radioactivity incorporated into the product of the enzymatic reaction was measured according to the "section (2) Analysis of enzymatic activity" above, placing the amount of the enzyme of the present invention at 1.3 µg, which contained the donor substrate (UDP- sugar; UDP-GlcUA or UDP-GalNAc) in a fixed concentration (240 µM) and adding to it the other radiolabeled donor substrate (UDP-sugar radiation; UDP- [3H] GalNAc or UDP- [14C] GlcUA) with various concentrations (0.6 to 200 µM). Independent tests were performed three times, and the average value was used as the measured value.

The relationship between the incorporated radioactivity (V) and the ratio between UDP-sugar substrate (S) is shown in Figure 9, and its double reciprocal representation in Figure 10. The black circle and the white square in the drawings show the results on UDP-GlcUA and UDP-GalNAc, respectively.

As a result, the Km for UDP-GlcUA and the Km for UDP-GalNAc were calculated to be 3.44 µM and 31.6 µM, respectively.

Sequence listing

<110> Seikagaku Corporation

<120> CONDROITIN-POLYMERASE AND DNA CODING IT

<150> JP 2001-244685

<151> 2001-08-10

<150> JP 2001-324127

<151> 2001-10-22

<150> JP 2002-103136

<151> 2002-04-05

<160> 11

<170> PatentIn Ver. 2.1

<210> 1

<211> 2058

<212> DNA

<213> Escherichia coli

<220>

<221> CDS

<222> (1) .. (2058)

<400> 1

image 1

Y

image2

image 1

image2

<210> 2

<211> 686

<212> PRT

<213> Escherichia coli

<400> 2

image3

image 1

image2

<210> 3 5 <211> 14483

<212> DNA

<213> Escherichia coli

<220> 10 <221> CDS

<222> (3787) .. (5847)

<400> 3

image4

image 1

image 1

image 1

image 1

image 1

image2

<210> 4 5 <211> 108

<212> DNA

<213> Artificial sequence

<220> 10 <221> CDS

<222> (1) .. (108)

<223> Description of the artificial sequence: sequence encoding the His-tag amino acid sequence

<400> 4 15

image2

twenty

<210> 6 <211> 27 <212> DNA <213> Artificial sequence

25

<220> <223> Description of the artificial sequence: primer sequence CS-S <400> 5

acccaacact gctacaacct atatcgg

27

30

<210> 6 <211> 30 <212> DNA <213> Artificial sequence

35

<220> <223> Description of the artificial sequence: primer sequence CS-AS

<400> 6

40

gcgtcttcac caataaatac ccacgaaact 30

<210> 7

<211> 26 <212> DNA <213> Artificial sequence

<220> <223> Description of the artificial sequence: TM-S primer sequence

<400> 7

cgagaaatac gaacacgctt tggtaa

26

<210> 8 <211> 28 <212> DNA <213> Artificial sequence

<220> <223> Description of the artificial sequence: TM-AS primer sequence

<400> 8

actcaatttt ctctttcagc tcttcttg

28

<210> 9 <211> 30 <212> DNA <213> Artificial sequence

<220> <223> Description of the artificial sequence: primer sequence K4C-SP

<400> 9

cgggatcccg atgagtattc ttaatcaagc

30

<210> 10 <211> 30 <212> DNA <213> Artificial sequence

<220> <223> Description of the artificial sequence: primer sequence K4C-AS

<400> 10

ggaattccgg ccagtctaca tgtttatcac

30

<210> 11 <211> 36 <212> PRT <213> Artificial sequence

<220> <223> Description of the artificial sequence: His-tag amino acid sequence

<400> 11

image2

Claims (16)

1. Isolated protein selected from the group consisting of the following sections (A) and (B):

(TO)

a protein comprising the amino acid sequence represented by the SEC. ID. No. 2;

(B)

a protein comprising the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity.

2.

Protein according to claim 1, wherein the protein is in soluble form.

3.

DNA comprising one of the following sections (a) to (c):

(to)

DNA encoding a protein consisting of the amino acid sequence represented by the SEC. ID. No. 2;

(b)

DNA encoding a protein consisting of the amino acid sequence in which 1 to 30 amino acid residue (s) in the amino acid sequence represented by SEC. ID. No. 2 are removed, replaced, inserted or transposed, and having chondroitin polymerase activity;

(C)

DNA that hybridizes to

(i)

the DNA in section (a), or

(ii)

a DNA complementary to the DNA in section (a) under severe conditions in which hybridization is performed at 42 ° C in a solution containing 50% formamide, 4 x SSC, 50 mM HEPES (pH 7.0), 10 x solution of Denhardt and 100 µg / ml of salmon sperm DNA, and that encodes a protein that has chondroitin polymerase activity.

Four.

DNA according to claim 3, wherein the DNA in section (a) is represented by the SEC. ID. No. 1

5.

Vector comprising the DNA of claim 3 or 4.

6.

Vector according to claim 5, which is an expression vector.

7.

Transformant in which a host is transformed with the vector of claim 5 or 6.

8.

Method for producing a chondroitin polymerase, which comprises: culturing the transformant of claim 7; Y collect chondroitin polymerase from the cultured material.

9.

Agent for the synthesis of the sugar chain, which comprises the protein of claim 1 or 2 and which has chondroitin polymerase activity.

10.

Process for producing a sugar chain represented by the following formula (3), comprising at least one step that allows the synthesizing agent of claim 9 to contact a GalNAc donor and a sugar chain represented by the formula ( 1) following:

GlcUA-X-R1 (1) GalNAc-GlcUA-X-R1 (3) in which GlcUA represents D-glucuronic acid; GalNAc represents N-acetyl-D-glucosamine; X represents GalNAc or GlcNAc in which GalNAc represents N-acetyl-D-glucosamine; -represents a glycosidic bond and R1 represents any group. 11. A method for producing a sugar chain represented by the following formula (4), comprising at least one step that allows the synthesizing agent of claim 9 to contact a GalNAc donor and a sugar chain represented by the formula (1) following: GlcUA-X-R1 (1) GlcNAc-GlcUA-X-R1 (4) in which GlcUA represents D-glucuronic acid; GalNAc represents N-acetyl-D-glucosamine; X represents GalNAc or GlcNAc in which GalNAc represents N-acetyl-D-glucosamine; -represents a glycosidic bond and R1 represents any group. 12. Method for producing a sugar chain represented by the following formula (5), comprising at least one step that allows the synthesizing agent of claim 9 to contact a GlcUA donor and a sugar chain represented by the formula (2) following: GalNAc-GlcUA-R2 (2) GlcUA-GalNAc-GlcUA-R2 (5) in which GlcUA represents D-glucuronic acid; GalNAc represents N-acetyl-D-glucosamine; X represents GalNAc or GlcNAc in which GlcNAc represents N-acetyl-D-glucosamine; -represents a glycosidic bond and R2 represents any group. 13. Method for producing a sugar chain selected from the following formulas (6) and (8), comprising at least one step that allows the synthesizing agent of claim 9 to contact a GalNAc donor, with a Donor of GlcUA and with a sugar chain represented by the following formula (1): GlcUA-X-R1 (1) (GlcUA-GalNAc) n-GlcUA-X-R1 (6) GalNAc- (GlcUA-GalNAc) n-GlcUA-X-R1 (8) in which GlcUA represents D-glucuronic acid; GalNAc represents N-acetyl-D-glucosamine; X represents GalNAc or GlcNAc in which GlcNAc represents N-acetyl-D-glucosamine; -represents a glycosidic bond; R1 represents any group; and n is an integer of 1 or more. 14. Method for producing a sugar chain selected from the following formulas (7) and (9), comprising at least one step that allows the synthesizing agent of claim 9 to contact a GalNAc donor, with a Donor of GlcUA and with a sugar chain represented by the following formula (2): GalNAc-GlcUA-R2 (2) (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (7) GlcUA- (GalNAc-GlcUA) n-GalNAc-GlcUA-R2 (9) in which GlcUA represents D-glucuronic acid; GalNAc represents N-acetyl-D-glucosamine; -represents a glycosidic bond; R2 represents any group; and n is an integer of 1 or more.

fifteen.

Hybridization probe comprising a nucleotide sequence complementary to the nucleotide sequence represented by SEC. ID. No. 1

16.

Glucosyl transfer catalyst comprising the protein of claim 1 or 2 and having chondroitin polymerase activity.