JP2002209586A - Dna encoding transmembrane domain-lacking type n- acetylglucosamine deacetylation enzyme - Google Patents

Abstract

PROBLEM TO BE SOLVED: To efficiently obtain an N-acetylglucosamine deacetylation enzyme. SOLUTION: This DNA encoding the partial polypeptide of the polypeptide of an N-acetylglucosamine N-deacetylation-N-sulfate group transfer enzyme is characterized in that the partial polypeptide obtained by inserting the DNA into an expression vector and then expressing the partial polypeptide does not have the N-terminal domain and C-terminal domain of the polypeptide of the N-acetylglucosamine N-deacetylation-N-sulfate group transfer enzyme in such an extent as capable of having an action for deacetylating the N- acetylglucosamine residue of a sugar chain having a heparin skeleton. A host cell is transformed with an expression vector containing the DNA, and the obtained transformant is cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA encoding N-acetylglucosamine deacetylase that can be expressed in a transformant.

[0002]

2. Description of the Related Art Heparin and heparan sulfate are a kind of sulfated glycosaminoglycan, and have anti-coagulant activity, smooth muscle growth inhibitory activity, specific binding activity with growth factors and cytokines, and cell growth / differentiation. It is responsible for a wide range of important biological functions such as regulation of wound healing and regulation of tissue formation. The basic skeleton is a disaccharide repeating structure of hexuronic acid and glucosamine. Specifically, the basic skeleton is usually a repeating structure in which a disaccharide composed of hexuronic acid α or β1-4 glucosamine is α1-4 bonded.

[0003] Heparin and heparan sulfate are usually prepared by extraction from animal organs and tissues, and have not been successfully produced by microorganisms.

In the biosynthesis of heparin and heparan sulfate, after a basic skeleton in which glucuronic acid and N-acetylglucosamine are bonded is formed, deacetylation / N-sulfation of N-acetylglucosamine residues, glucuronic acid residues Undergoes a modification such as C5 epimerization (iduron oxidation of glucuronic acid), 2-O sulfation of glucuronic acid or iduronic acid residue, 6-O sulfation of glucosamine residue, and 3-O sulfation.

In such biosynthesis of heparin and heparan sulfate, N-deacetylation and N-sulfation of N-acetylglucosamine residues are the first steps of the modification reaction, and if this reaction does not take place, other reactions will take place. No modification reaction is expected to occur. Therefore, it can be said that the steps of deacetylation and N-sulfation are essential processes in artificial heparin and heparan sulfate synthesis.

[0006] The enzyme which catalyzes N-deacetylation and N-sulfation is expressed as a single molecule having two kinds of enzymatic activities as far as it is known. It is named N-deacetylated N-sulfotransferase (hereinafter also referred to as "NDST"). The structure of this enzyme is a type II membrane protein like other glycosaminoglycan sulfotransferases, and a transmembrane domain in which hydrophobic amino acids are gathered in the region near the amino terminus (hereinafter also referred to as "N-terminus"). There is. The carboxyl terminal (hereinafter also referred to as "C-terminal") side is N-sulfotransferase (hereinafter "NS
T) is known to be an active moiety, and has two binding domains (5′-phosphosulfate binding domain and 5′-phosphosulfate binding domain) specific for active sulfate (PAPS), which is a sulfate group donating substrate.
3'-phosphate binding domain) (Sueyoshi
T., et al. (1998) FEBS Lett. 433, 211-4). It is thought that an N-deacetylase (hereinafter also referred to as "NDA") active portion is present on the N-terminal side, and it is said that there is a common structure of deacetylase (Berninsone).
P., et al. (1998) J. Biol. Chem. 273, 25556-9).

[0007] Four types of NDST have been found to date, and NDST-1 (rat (rNDST-1); Hashimoto Y., et al.
(1992) J. Biol. Chem. 267, 15744-15750, human (hNDS
T-1); Dixon J., et al. (1995) Genomics 26, 239-24
4), NDST-2 (Orellana A., etal. (1994) J. Biol. C
hem. 269, 2270-6, Toma L., et al. (1998) J. Biol.
Chem. 273, 22458-65), NDST-3 (Aikawa J., et al.
(1999) J. Biol. Chem. 274, 2690-5), NDST-4 (Aikawa
J., et al. (1999) Glycoconj. J. 16, S40), etc. (these are collectively referred to simply as "NDST" below). Regarding the expression of recombinant proteins of these enzymes by genetic engineering, expression of all structures in animal cells has already been reported (for example, Cheung WF, et al. (199).
6) Biochemistry 35, 5250-6, Pikas DS, et al. (2
000) Biochemistry 39, 4552-8). However, when expressed in animal cells, the production amount is low and the production cost is high, which is not suitable for industrial production.

[0008] Therefore, an attempt has been made to introduce an NDST gene into a highly productive microorganism such as Escherichia coli to produce an enzyme (Sueyoshi T., et al. (1998) FEBS Lette).
r 433, 211-4, Kakuta Y., et al. (1999) J. Biol. C
274, 10673-6), only the N-sulfotransferase active portion on the C-terminal side was successfully expressed, and the expression of the entire structure was impossible. Therefore, an attempt has been made to express a N-terminal N-deacetylase activity region as a fusion protein, but the expression of the active protein has not been successful (Berninsone P., et a
l. (1998) J. Biol. Chem. 273, 25556-9), it is believed that expression of a region thought to be responsible for N-deacetylase activity in microorganisms is impossible.

[0009]

Therefore, the expression of NDST and the expression of the region responsible for NDA activity alone, which are required for the preparation of heparin and heparan sulfate by genetic recombination on an industrial scale, are still successful. I haven't. Therefore, an object of the present invention is to obtain an NDA efficiently.

[0010]

Means for Solving the Problems The present inventors have conducted intensive studies to express the NDA activity necessary for efficiently synthesizing heparin and heparan sulfate in microorganisms.
Identify the region of the DNA encoding each of the N-deacetylase active region and N-sulfonase active region of DST, and further include the N-terminal transmembrane region and the C-terminal N-sulfation of NDST By removing the region containing the enzyme active region and ligating it with an appropriate microorganism expression vector, it was found that a recombinant protein maintaining NDA activity was expressed in microorganisms, and thus completed the present invention.

That is, the gist of the present invention is as follows. (1) DNA encoding a partial polypeptide of N-acetylglucosamine N-deacetylated / N-sulfotransferase polypeptide, which is obtained by inserting the DNA into an expression vector and expressing the DNA. N-acetylglucosamine N-deacetylated / N-sulfotransferase polypeptide to the extent that the partial peptide can have the effect of deacetylating the N-acetylglucosamine residue of the sugar chain having a heparin skeleton. DNA having no amino terminal region and no carboxyl terminal region. (2) DNA of 2 kbp or less that can hybridize under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 3 or 6 or a nucleotide sequence complementary thereto, and the N of sugar chain having a heparin skeleton -DNA encoding a polypeptide having an action of deacetylating acetylglucosamine residues. (3) An expression vector containing the DNA according to (1) or (2). (4) The expression vector according to (3), further comprising a DNA encoding a polypeptide having an activity of transferring a sulfate group to an amino group of a glucosamine residue. (5) A transformant obtained by transforming a host cell with the expression vector according to (3) or (4). (6) The transformant according to (5), wherein the host cell is a microorganism. (7) The transformant according to (6), wherein the microorganism is Escherichia coli. (8) An enzyme obtainable by growing the transformant according to any one of (5) to (7), which deacetylates an N-acetylglucosamine residue of a sugar chain having a heparin skeleton. N-acetylglucosamine deacetylase lacking a transmembrane region having an action. (9) A composition comprising the N-acetylglucosamine deacetylase lacking the transmembrane region of (8) and an N-sulfotransferase.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in more detail with reference to embodiments.

<1> DNA of the Present Invention The DNA of the present invention is a DNA encoding a partial polypeptide of the NDST polypeptide, which is obtained by inserting the DNA into an expression vector and expressing it. Is a DNA that does not have the N-terminal region and the C-terminal region of the NDST polypeptide to the extent that it can have the effect of deacetylating the N-acetylglucosamine residue of the sugar chain having a heparin skeleton. is there.

N-deacetylated N-sulfotransferase is
An enzyme that catalyzes both N-deacetylation and N-sulfation reactions of N-acetylglucosamine residues in sugar chains having a heparin skeleton, and consists of a single polypeptide showing two types of enzymatic activities Means

In the present specification, the heparin skeleton refers to a disaccharide in which hexuronic acid and glucosamine are linked by α or β 1,4 glycoside as a basic unit. Finger.
The partial polypeptide encoded by the DNA of the present invention has an effect of deacetylating the N-acetylglucosamine residue constituting the heparin skeleton (in the present specification, “NDA activity”).
Also referred to as).

The expression "having NDA activity" means that when a DNA encoding a partial polypeptide is inserted into an expression vector and expressed in a host cell (preferably a microbial cell), the NDA activity in an extract of the host cell is increased. It means that the activity is detected, and more specifically, when the NDA activity is measured according to the method described in Examples described later, the activity is detected.

The DNA of the present invention encodes an N-deacetylated / N-sulfotransferase (NDST) polypeptide lacking the N-terminal region and the C-terminal region. The N-terminal region and the C-terminal region are regions where the polypeptide obtained by expression has NDA activity in the absence of them. The partial polypeptide of NDST having such activity is hereinafter referred to as "the polypeptide of the present invention".

The N-terminal region is, for example, the N-terminal transmembrane region of the NDST polypeptide and the N-terminal region including a portion from the region to the N-terminal. N-terminal region, as long as the polypeptide of the present invention obtained by expression has NDA activity
A region on the C-terminal side of the transmembrane region may be included.

In the present invention, the N-terminal transmembrane region is defined by the commonly used hydropathy plot of J. Kyte and RF Doolittle (J. Mol. Biol. 157 (1982), 105-132). When the amino acid sequence of the NDST was analyzed (an example of the results of analysis of NDSTs derived from humans and rats by hydropathy plots is shown in FIG. 1), it is a region consisting of 20 or more amino acids recognized as a hydrophobic region. In general, a region existing near the N-terminus of a type II membrane protein is referred to (a region indicated by an arrow in FIG. 1).

The C-terminal region is usually a region that is responsible for the N-sulfotransferase (NST) activity of the NDST polypeptide and a region from that region to the C-terminal of the NDST polypeptide. The C-terminal region may be longer or shorter as long as the polypeptide of the present invention obtained by expression has NDA activity.

The deletion of the N-terminus and the C-terminus of the translated NDST polypeptide to any length can be carried out by ordinary genetic recombination techniques. , It is easy for a person skilled in the art to identify appropriate N-terminal and C-terminal regions.

The translated region of the DNA of the present invention is 2000 b
p (2 kbp) or less, more preferably 1
It is 700 bp or less, most preferably 1600 bp or less.

A preferred example of such a DNA of the present invention is, for example, a DNA consisting of base numbers 567 to 1882 or 567 to 2116 of the rat base sequence described in SEQ ID NO: 1, or a DNA described in SEQ ID NO: 4. Examples include DNAs consisting of base numbers 337 to 1653 or 337 to 1887 in the human base sequence.

As long as the DNA of the present invention encodes the polypeptide of the present invention exhibiting NDA activity, the nucleotide sequence of the DNA of NDA (preferably the nucleotide sequence of SEQ ID NO: 3 or 6 or the nucleotide sequence of SEQ ID NO: 3 or 6) DNA having a nucleotide sequence of 2 kbp or less having a nucleotide sequence capable of hybridizing under stringent conditions to a nucleotide sequence consisting of 1317) or a nucleotide sequence complementary thereto. Such DNA also does not have a sequence corresponding to the N-terminal region described above.

The above-mentioned stringent conditions are as follows.
In general, the conditions used for the nucleic acid detection method by the hybridization method are referred to, for example, 37.5% formamide, 5 × SSPE (sodium chloride / sodium phosphate / EDT).
A (ethylenediaminetetraacetic acid) buffer), 5 × Denhardt's solution, and conditions of 42 ° C. in the presence of 0.5% SDS (sodium dodecyl sulfate) are exemplified.

The polypeptide of the present invention encoded by the DNA of the present invention may be prepared as a fusion protein fused with another polypeptide as long as the NDA activity is not impaired.

Since the polypeptide of the present invention encoded by the DNA of the present invention has NDA activity and lacks a transmembrane region, it is also referred to as a transmembrane region-deficient N-acetylglucosamine deacetylase.

<2> Vector of the Present Invention The vector of the present invention is an expression vector containing the above-described DNA of the present invention.

The vector of the present invention is a vector obtained by introducing the DNA of the present invention into an appropriate expression vector (such as a phage vector or a plasmid vector) capable of expressing the DNA of the present invention. As the above-mentioned expression vector, the DN of the present invention
Those suitable for the host cell in which A is expressed are selected. Such host-vector systems include COS cells (eg, COS
Mammalian cells such as OS-1 cells, COS-7 cells) and 3LL-HK46 cells, and pEF-BOS, pCXN2 (Niwa, H., Yamanura, K. and Mi
yazaki, J. (1991) Gene 108, 193-200), pCMV-2 (Eastman Kodak), pCEV18, pME1
8S (Maruyama et al., Med. Immunol., 20, 27 (1990)) or pSVL
(Pharmacia Biotech) and other expression vectors for mammalian cells, E. coli, pTr
cHis (Invitrogen), pGEX (Pharmacia Biotech), pTrc99 (Pharmacia Biotech), pKK233-3 (Pharmacia Biotech), pEZZZ18 (Pharmacia Biotech), pC
H110 (Pharmacia Biotech), pET (Stratagene), pBAD (Invitrogen), pRSET
(Invitrogen) and pSE420 (Invitrogen) in combination with an expression vector for prokaryotic cells. In addition, insect cells, yeast,
Bacillus subtilis and the like, and various vectors corresponding thereto. In particular, prokaryotic cells such as Escherichia coli are preferable as host cells, and phage vectors and plasmid vectors capable of introducing the DNA of the present invention into such host cells are preferable as vectors.

It is preferable that the vector of the present invention has the DNA of the present invention inserted in a direction showing the activity as NDA when the DNA of the present invention is transcribed and translated into a polypeptide. It is also possible to construct the vector of the present invention so that the polypeptide of the present invention encoded by the DNA of the present invention is expressed as various fusion proteins. It is preferable to select a vector that does not allow expression of a fusion protein with a polypeptide having activity. When the vector of the present invention is constructed so as to express a fusion protein of the polypeptide of the present invention, the fusion protein comprises the polypeptide of the present invention encoded by the DNA of the present invention and various tag peptides (for example, His, FLAG, Protein A). , CBP (Calmoduli
n Binding Protein), GST (Glutathione S-Transferas)
e) etc.).

The vector of the present invention includes the above-described vector of the present invention.
In addition to DNA, encodes a polypeptide having NST activity
DNA (hereinafter referred to as NST-DNA) may be further contained, in which case, the NST-DNA and the DNA of the present invention are so expressed that the polypeptide of the present invention and the sulfotransferase are not expressed as a fusion protein as described above, It is necessary to construct the vector of the present invention so that it is expressed as a separate protein. Using the vector of the present invention, a transformant described below is prepared and propagated to prepare a composition of the enzyme of the present invention described below and NST (composition of the present invention). It is.

According to the composition of the present invention, N-sulfated heparosan can be obtained, for example, by deacetylating N-acetylheparosan and performing N-sulfation.

The above NST-DNA is preferably SEQ ID NO: 1.
DNA having a base sequence consisting of base numbers 1884 to 3095 or 2118 to 3095, or base numbers 1654 to 286 of SEQ ID NO: 4
A DNA having a base sequence consisting of 5 or 1888 to 2865 is exemplified.

<3> Transformant of the Present Invention The transformant of the present invention is a transformant obtained by transforming a host cell with the expression vector of the present invention, that is, at least the recombinant of the present invention, It is a transformant that expresses a peptide.

The host cells may be derived from mammals, birds, reptiles, crustaceans, insects, and the like, in addition to microorganisms. Microorganisms are particularly preferred because of their high growth rate and ease of culture. It is preferably E. coli.

The transformant of the present invention can be easily prepared by transforming a host cell with the vector of the present invention according to a conventional method.

The polypeptide of the present invention can be expressed in a large amount by growing the transformant of the present invention. This growth can be performed in a culture solution or the like depending on the transformant, but the cells may be grown in vivo.

The case where the transformant of the present invention is propagated in a medium such as a culture solution is referred to as culturing. The culture obtained by such culturing is capable of expressing the transformant of the present invention and expressing the DNA of the present invention. Can be obtained by culturing the cells under suitable conditions. The enzyme of the present invention can be obtained from a culture (usually, a culture medium and cells after culturing) by combining methods generally used for purification of the enzyme.

<4> Enzyme of the Present Invention The enzyme of the present invention is an enzyme which can be obtained by growing the transformant of the present invention, and has an action of deacetylating an N-acetylglucosamine residue of a sugar chain having a heparin skeleton. N-acetylglucosamine deacetylase lacking a transmembrane region.

The enzyme of the present invention contains the polypeptide of the present invention encoded by the DNA of the present invention, and usually comprises a known ND.
A structure in which the N-terminal region and the C-terminal region have been deleted from ST,
For example, the NDA region of a known NDST,
This enzyme has a structure in which the terminal transmembrane region and the N-terminal intracellular region have been deleted. Therefore, the enzyme of the present invention is a known NDST
Unlike NST, it does not have NST activity.

The amino acid sequence of the polypeptide of the enzyme of the present invention (namely, the polypeptide of the present invention) is the amino acid number 41 to 479 or 41 to 557 of the rat NDST polypeptide shown in SEQ ID NO: 2, or the sequence thereof. A polypeptide consisting of amino acid Nos. 41 to 479 or 41 to 557 of the human NDST polypeptide represented by No. 5 is preferably exemplified, but as long as it has the above-mentioned enzymatic characteristics and structural characteristics, There is no limitation.

<5> Method for Preparing DNA of the Present Invention, etc. The DNA of the present invention, the expression vector of the present invention, the transformant of the present invention, and the enzyme of the present invention can be prepared, for example, by the following preparation methods.

(1) Preparation of Total RNA The DNA of the present invention can be prepared from total RNA of cells expressing NDST, preferably mammalian cells. As such cells, for example, animal tissues such as liver and brain are suitable. These tissues are preferably mammalian tissues, and more preferably human, dog, cat, sheep, cow, horse, goat, pig, mouse, rat, and guinea pig, but are not particularly limited.

The preparation of total RNA can be carried out according to a conventional method such as a method using a triazole reagent or the like, a guanidine / hot phenol method or the like.

(2) Selective amplification of the DNA of the present invention from total RNA The DNA of the present invention can be selectively amplified by the RT-PCR method or the like using the total RNA obtained in (1). RT-P total RNA
Although it may be used for amplification by the CR method, only mRNA may be previously purified using an oligo dT column or the like and used for the RT-PCR method.

Specifically, it was synthesized by a reverse transcription reaction.
Using the cDNA, the DNA of the present invention can be obtained by a conventional method such as PCR.
Can be prepared. When the DNA of the present invention is amplified by the PCR method using the above cDNA as a template, the desired present invention D
It is preferable to use a primer having a base sequence at both ends of NA or a base sequence complementary thereto. Preferred examples of such primers include those having the nucleotide sequence of SEQ ID NO: 8 (primer 2 for rat) or SEQ ID NO: 14 (primer 2 for human), SEQ ID NO: 12 (primer 6 for rat), and SEQ ID NO: 18. (Primer 6 for human), SEQ ID NO: 19 (primer 7 for human) or SEQ ID NO: 13 (primer 7 for rat). The DNA of the present invention thus specifically amplified can be fractionated by, for example, gel electrophoresis or other sorting means based on molecular size, electric charge, or the like, and the desired fraction can be collected according to a conventional method.

(3) Preparation of the Vector of the Present Invention from the DNA of the Present Invention The DNA of the present invention obtained above is inserted into an appropriate vector suitable for a host cell according to a conventional method. For example, when introducing the DNA of the present invention into Escherichia coli, the DNA of the present invention is inserted into an expression vector that can be expressed in Escherichia coli. In the case of the E. coli TOP10 strain, an expression vector such as pTrcHis is exemplified.

(4) Preparation of the Enzyme of the Present Invention from a Host Cell The above-described vector of the present invention can be introduced into a host cell according to a conventional method. For example, when the enzyme of the present invention is prepared using the vector of the present invention in which the present invention has been introduced into pTrcHis, for example, Escherichia coli TOP10 is transformed with the vector of the present invention, and then a medium containing ampicillin (eg, LB
Medium), for example, at 37 ° C. for 5 hours, and then isopropylthio-β-D-galactoside (IPTG) is added, followed by further culturing under appropriate conditions (eg, 37 ° C. for 3 hours). After the culture solution is separated and centrifuged to collect the cells, the cells are washed with a phosphate buffer by a conventional method, and the cells are disrupted by, for example, a freeze-thawing method. It is possible to purify the enzyme of the present invention from the cell lysate. In the embodiment of the method for preparing the enzyme of the present invention using the vector of the present invention created using pTrcHis, the enzyme of the present invention is a tag peptide, Hi.
Since expressed as a fusion protein with the s tag, for example, the enzyme of the present invention can be easily purified by a method such as affinity chromatography using magnetic nickel agarose beads as a carrier,
By using an anti-His antibody, the enzyme of the present invention can be easily identified.

[0049]

EXAMPLES The present invention will be described more specifically with reference to the following examples.

<1> Preparation of DNA Fragment and Expression Plasmid Rat liver was collected, immediately frozen with liquid nitrogen and homogenized, and then total RNA was extracted by a method using a triazole reagent (manufactured by Gibco BRL). From this total RNA, oligo (dT) -cel
mRNA using a lulose column (Pharmacia)
Was purified. This rat liver-derived mRNA, oligo (dT) 12-1
8 or random primer and reverse transcriptase (Gibco B
RL) and a reverse transcription reaction at 55 ° C. for 60 minutes, followed by ribonuclease (RNase) H (Gibco BRL).
The NA was cleaved off to prepare cDNA.

Using the cDNA obtained above as a template, pfu-DN
Table 1 using A polymerase (Stratagene)
The PCR method was performed by combining the sense primer and the antisense primer shown in (1), and a DNA fragment having a HindIII cleavage sequence at each end was amplified. The PCR reaction is
First, denaturation at 94 ° C for 1 minute, denaturation at 94 ° C for 45 seconds, and
Twenty-five cycles of amplification reaction consisting of annealing for 30 seconds and extension at 74 ° C. for 3 minutes were performed, and finally, the conditions were such that extension was performed at 74 ° C. for 10 minutes. After subjecting this PCR reaction solution to 1.2% agarose gel electrophoresis, use Gel Extraction Kit (QIAG
(Manufactured by EN)) to prepare a PCR product containing each NDST DNA fragment (Table 1).

[0052]

[Table 1]

When reverse transcription PCR is similarly performed on human-derived NDST (the nucleotide sequence encoding human-derived NDST is shown in SEQ ID NO: 4), for example, all the DNA extracted from tissues such as human brain and liver can be used. Using RNA or mRNA, primer 1 is SEQ ID NO: 14, primer 2 is SEQ ID NO: 15, primer 3
Can be performed in the same manner by changing primer No. 16 to SEQ ID No. 17, primer 6 to SEQ ID No. 19, and primer 7 to a primer having the nucleotide sequence shown in SEQ ID NO.

Each PCR product was treated with the restriction enzyme HindIII to prepare a DNA fragment for insertion. E. coli expression vector p
TrcHis (manufactured by Invitrogen) is treated with the restriction enzyme HindIII, and further treated with calf small intestine-derived alkaline phosphatase (CIAP; manufactured by NEB).
The fragment was ligated (16 ° C., 16 hours) using T4 DNA ligase (manufactured by NEB), and E. coli competent cells (TOP10
And transformed, and cloned a positive colony resistant to ampicillin. The plasmid was purified from the cloned Escherichia coli, subjected to PCR, restriction enzyme treatment, and nucleotide sequence analysis.The inserted DNA fragment was in the forward direction, and the target expression plasmid containing the correct protein expression codon frame was introduced. E. coli clones were selected.

[0055]

[Table 2]

<2> Expression of recombinant protein E. coli having each expression plasmid cloned in <1> was transformed into an LB medium (50 μg / ml) containing ampicillin (50 μg / ml).
and isopropylthio-β-D-
Galactoside (IPTG) was added to a final concentration of 1 mM, and the mixture was further cultured at 37 ° C. for 3 hours to induce the expression of each recombinant protein. Take a part (1.5 ml) of the culture solution, collect the cells by centrifugation, and add 20 mM phosphate buffer (pH
7) The cell suspension obtained by repetition of freeze-thaw by suspending in 100 μl and alternately pouring into liquid nitrogen and a water bath at 40 ° C. three times was subjected to 10% SDS polyacrylamide gel electrophoresis, followed by nitrocellulose. Western blotting was performed using the membrane. Since the expressed recombinant protein is a fusion protein having a His tag consisting of histidine of 6 residues, it can be confirmed by staining using an anti-His antibody (manufactured by QIAGEN).

[0057]

[Table 3]

Among the expressed proteins, His-A, His-B, Hi
For sC and His-D, substantially no band was detected at the expected molecular weight position. This may be due to low expression, degradation, or extreme insolubilization.His-E, His-F, His-G, and His-H appear to have been degraded because a band appeared. It is unlikely that His-B, which has no transmembrane region, was extremely insoluble because no band was detected. From these results, fusion polypeptides containing the amino acid sequences of both NDA and NST polypeptides and NDA polypeptides having a transmembrane region may not be suitable for the purpose of preparing large quantities of active enzymes. found. However, for the expressed proteins His-E, His-F, His-G and His-H, a clear anti-His antibody positive band was observed at the expected molecular weight position, indicating that the target recombinant protein was produced. Do you get it.

<3> Construction of Escherichia coli Expression Vectors for NDA and NST There is a principle of incompatibility that only one kind of plasmid of the same type (having the same origin of replication) is retained in one cell. Basic technology (1996) edited by Michio Ota, published by Nane; p23). To simultaneously express both enzyme activities in the same Escherichia coli, use two types of plasmids having different replication origins or two types that can be translated into one plasmid. It is conceivable to insert DNA sequences encoding the above proteins side by side so as to express different polypeptides. In this example, the latter method was studied.

Using plasmid pTrcHis-E as a template, primer 8 (base sequence is shown in SEQ ID NO: 20) and primer 9
(Base sequence is shown in SEQ ID NO: 21), and D corresponding to His-E having a restriction enzyme NcoI cleavage sequence at both ends by PCR.
The NA fragment was amplified. In the PCR reaction, pfu-DNA polymerase (manufactured by Stratagene) was used as a synthase, followed by denaturation at 94 ° C. for 1 minute,
Perform 10 cycles of amplification reaction consisting of annealing at 55 ° C for 30 seconds and extension at 74 ° C for 1 minute and 50 seconds, followed by denaturation at 94 ° C for 45 seconds, annealing at 58 ° C for 30 seconds and 74 ° C for 1 minute 5 minutes.
Perform 20 cycles of amplification reaction consisting of 0 seconds extension, and finally
The extension was performed at 4 ° C. for 10 minutes. After subjecting the PCR reaction solution to 1.2% agarose gel electrophoresis, Gel Ext
raction Kit (QIAGEN), and purify each NDST
A PCR product containing the NA fragment was prepared. Further, the DNA fragment was treated with NcoI to prepare a DNA fragment for insertion.

The plasmid pTrcHis-H (2 μg) was replaced with NcoI (50 U).
For 3 hours at 37 ° C., and then treated with alkaline phosphatase derived from calf small intestine (CIAP; manufactured by NEB). The opened plasmid was ligated with the above restriction enzyme-treated DNA fragment using T4 DNA ligase (manufactured by NEB) (16).
℃, 16 hours), and competent E. coli cells (TOP10 strain)
And transformed, and a positive colony resistant to ampicillin was cloned. The plasmid is purified from the cloned Escherichia coli, subjected to PCR, restriction enzyme treatment, and nucleotide sequence analysis. The target expression plasmid (pTrcHis-E: His-H)
Was introduced into E. coli. The primer 9 has a sequence corresponding to the SD sequence that is a ribosome binding site, and was designed so that the SD sequence also exists immediately upstream of the translation sequence corresponding to His-H in the obtained expression plasmid. ing.

E. coli having the obtained expression plasmid was
The recombinant protein expression was induced by the same operation as in <2>, and Western blotting was performed.
(64 kDa) and bands corresponding to His-H (43 kDa), indicating that both proteins (His-E and His-H) were expressed.

<4> Measurement of enzyme activity The recombinant protein-derived Escherichia coli culture obtained in <2> and <3> was centrifuged to separate cells, and 2 ml of an enzyme buffer (10 mM TrisHCl pH 7.5, 0.1% Triton X100, 10 mM
MgCl ₂ , 2 mM CaCl ₂ , 20% glycerol, 50 mM NaCl)
And sonicate three times for 30 seconds under ice cooling to destroy the cells,
A cell suspension was prepared. These were used as enzyme solutions.
CDS for NST activity as an acceptor substrate
(Total desulfation) -Heparin (Seikagaku Corporation)
K5 (N-acetylheparosan; capsular polysaccharide prepared from Escherichia coli K5 strain) was used for coupling activity of NDA activity and NST activity at 50 μg / vial. Donor substrates include [
³⁵ S] -PAPS (3'-phosphoadenosine 5'-phosphosulfate; NEN), 1 nmol (about 1.6 μCi) / vial, and 50 mM HEP
In a buffer solution containing ES (pH 7), 0.15 M NaCl, 0.75 mg / ml protamine hydrochloride (total volume 50 μl / vial), 5 to 10 μl of the above enzyme solution was used. The enzyme reaction was carried out at 37 ° C. for 20 minutes. The reaction solution was precipitated with ethanol, suspended in 20 μl of distilled water, spotted on filter paper (Toyo Filter Paper No. 50), and ethanol:
M Performed paper filter chromatography using a mixed solution of ammonium acetate (65:35 (v / v)) as a developing solvent (room temperature for 2 days),
The amount of sulfate groups transferred from PS to the polymer acceptor substrate was determined by measuring the radioactivity of [ ³⁵ S] -sulfuric acid remaining at the origin. Table 4 shows the results.

[0064]

[Table 4]

From the above, His-G and His-H have NST activity, and coupling activity appears in combination with them, indicating that His-E and His-F have NDA activity. In addition, it was found that an enzyme solution derived from Escherichia coli (His-E: His-H) expressing both His-E and His-H as separate polypeptides has both NDA activity and NST activity.

[0066]

Industrial Applicability According to the present invention, a DNA encoding NDA and a vector containing the same, which can be expressed even in microorganisms, are provided, and NDA can be prepared in large quantities.

[0067]

[Sequence List] SEQUENCE LISTING <110> Seikagaku Corporation <120> DNA encoding N-acetylglucosamine deacetylase lacking the transmembrane region <130> P-8153 <160> 21 <210> 1 <211> 4051 <212> DNA <213> Rattus sp. <220> <221> CDS <222> (447) .. (3095) <400> 1 gaattccaag acagaatatc agtcctccct ccgtttccca ggtcagctcc ctagccttta 60 ccctgtgggc ccaacgtgaa gagcgtgaag agcgcggcat gcagcacccc ctggctggcc 120 cggtgtcttt tgacctgctg ttgactgctc ccaggaggcc ctgacgccct ggggcacttt 180 tgctctgcac aggaccacgt tggggtttgc catggtgaca taaaggggtg tagaggaagg 240 aaggagcaag accagcctgt agactgtgcc cctggctggg cgaaagggcc aggggcccgg 300 agccttctct ccactgccct ctgaacgtct cacttcctaa gtctctgcat ttctcagtca 360 gtggacaact tttggaactc ctgtgtgagg acttcggggg accaggccag gctgggcctc 420 tgctggttga gatctcgggg gccaga atg cct gcc ctg gcg tgc ctc cgg agg 473 Met Pro Ala Leu Ala Cys Leu Arg Arg 15 ctg tgt cgg cac ctg tcc cca cag gct gt c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c ct c gt c Pro Gln Ala Val Leu Phe Leu Leu Phe Val 10 15 20 25 ttc tgc ctg ttc agc gtg ttt gtc tcg gcc tac tac cta tat ggt tgg 569 Phe Cys Leu Phe Ser Val Phe Val Ser Ala Tyr Tyr Leu Tyr Gly Trp 30 35 40 aac cgg ggc ctc gag ccc tcg gca gat gct tct gag tcc gac tgc ggg 617 Asn Arg Gly Leu Glu Pro Ser Ala Asp Ala S er Glu Ser Asp Cys Gly 45 50 55 gac cca cca cct gtc gcc cct agc cgt ctc ctg cca atc aag cct gtg 665 Asp Pro Pro Pro Val Ala Pro Ser Arg Leu Leu Pro Ile Lys Pro Val 60 65 70 cag gcg gtc gcc cct tct cga aca gac ccg ctg gtg ctg gta ttt gtg 713 Gln Ala Val Ala Pro Ser Arg Thr Asp Pro Leu Val Leu Val Phe Val 75 80 85 gag agc ctc tat tca cag ctg ggc cag gag gtg gtg gcc atc ctg gaa 76 Leu Tyr Ser Gln Leu Gly Gln Glu Val Val Ala Ile Leu Glu 90 95 100 105 tcc agt cgc ttc aag tac cga aca gaa att gca ccg ggg aag ggg gac 809 Ser Ser Arg Phe Lys Tyr Arg Thr Glu Ile Ala Pro Gly Lys Gly Asp 110 115 120 atg ccc aca ctc aca gac aag ggc cga ggc cgc ttc gcc ctc atc atc 857 Met Pro Thr Leu Thr Asp Lys Gly Arg Gly Arg Phe Ala Leu Ile Ile 125 130 135 tat gag aac atc ctc aag tat gtc aac gat gcc tgg aac cgg gag 905 Tyr Glu Asn Ile Leu Lys Tyr Val Asn Leu Asp Ala Trp Asn Arg Glu 140 145 150 ctg ctg gac aag tac tgt gtg gcc tac ggc gtg ggc atc att ggc ttc 953 Leu Leu Cyp Ala Tyr Gly Val Gly Ile Ile Gly Phe 155 160 165 ttc aag gcc aat gag aac agc ctg ctg agt gca cag ctc aaa ggc ttc 1001 Phe Lys Ala Asn Glu Asn Ser Leu Leu Ser Ala Gln Leu Lys Gly Phe 170 175 180 185cct ctt cat tcg aac ctg ggc ttg aaa gac tgc agc atc aac 1049 Pro Leu Phe Leu His Ser Asn Leu Gly Leu Lys Asp Cys Ser Ile Asn 190 195 200 ccc aag tcc cca ctg ctg tac gtg aca cgg ccc agt gag gag gag aaa Lys Ser Pro Leu Leu Tyr Val Thr Arg Pro Ser Glu Val Glu Lys 205 210 215 ggt gtg ctg ccc gga gag gac tgg acg gtg ttc cag tct aac cac tct 1145 Gly Val Leu Pro Gly Glu Asp Trp Thr Val Val Phe Gln Ser Asn His Ser 220 225 230 acc tat gag cca gtg ctg ctg gcc aag acg cgc tcc tct gag tcc atc 1193 Thr Tyr Glu Pro Val Leu Leu Ala Lys Thr Arg Ser Ser Glu Ser Ile 235 240 245 cca cac ctg ggc gca gat gcc ggc ctg cat gct gcc ctg cac gct act 1241 Pro His Leu Gly Ala Asp Ala Gly Leu His Ala Ala Leu His Ala Thr 250 255 260 265 gtg gtc cag gac ctg ggc ctc cat gac ggc att cag cgt gtg ctg ttt 1289 Val Val Gln Asp L eu Gly Leu His Asp Gly Ile Gln Arg Val Leu Phe 270 275 280 ggc aac aac ctc aac ttt tgg ctg cat aag ctc gtc ttc gtg gac gct 1337 Gly Asn Asn Leu Asn Phe Trp Leu His Lys Leu Val Phe Val Asp Ala 295 gtg gcc ttc ctc aca ggg aag cgc ctc tca ctg cct ttg gac cga tac 1385 Val Ala Phe Leu Thr Gly Lys Arg Leu Ser Leu Pro Leu Asp Arg Tyr 300 305 310 atc ctg gtg gac att gat gag att ttt gta ggc aca cgc 1433 Ile Leu Val Asp Ile Asp Asp Ile Phe Val Gly Lys Glu Gly Thr Arg 315 320 325 atg aag gtg gag gat gtg aag gcc ctg ttt gat aca cag aat gaa ctt 1481 Met Lys Val Glu Asp Val Lys Ala Leu Asp Thr Gln Asn Glu Leu 330 335 340 345 cgt aca cat atc cca aac ttc acc ttc aac ctg ggc tac tca ggg aaa 1529 Arg Thr His Ile Pro Asn Phe Thr Phe Asn Leu Gly Tyr Ser Gly Lys 350 355 360 ttc ttc cac aca ggt acc gat gct gag gat gct ggg gac gac ctg ctg 1577 Phe Phe His Thr Gly Thr Asp Ala Glu Asp Ala Gly Asp Asp Leu Leu 365 370 375 ctg tcc tat gtg aaa gag ttc tgg tgg ttc ccc cac atg tgg agc cat 5 Leu Ser Tyr Val Lys Glu Phe Trp Trp Phe Pro His Met Trp Ser His 380 385 390 atg caa ccc cac ctc ttc cac aac cag tct gtg ctg gct gag cag atg 1673 Met Gln Pro His Leu Phe His Asn Gln Ser Val Leu Ala Glu Gln Met 395 400 405 gcc ctg aac aag aag ttc gct gtc gag cac ggc att ccc aca gat atg 1721 Ala Leu Asn Lys Lys Phe Ala Val Glu His Gly Ile Pro Thraspp Met 410 415 420 425 ggg tat gca gtg gca ccc cac tct ggt gtg tac cct gtg cat gtg 1769 Gly Tyr Ala Val Ala Pro His His Ser Gly Val Tyr Pro Val His Val 430 435 440 cag ctg tat gag gcc tgg aag caa gtg tgg aac atc cgt gtg acc agc 1817 Gln Leu Tyr Glu Ala Trp Lys Gln Val Trp Asn Ile Arg Val Thr Ser 445 450 455 aca gag gag tac ccg cat ctg aag cct gcc cgt tac cgc cgt ggc ttc 1865 Thr Glu Glu Tyr Pro His Leu Lys Pro Ala Arg Tyr Arg Arg Gly Phe 460 465 470 atc cac aat ggc atc atg gtc ctc cct cgg cag acc tgt ggt ctc ttt 1913 Ile His Asn Gly Ile Met Val Leu Pro Arg Gln Thr Cys Gly Leu Phe 475 480 485 aca cac acc atc ttc tac aac gag tac cct gga gg tcc agt gag ctg 1961 Thr His Thr Ile Phe Tyr Asn Glu Tyr Pro Gly Gly Ser Ser Glu Leu 490 495 500 505 gac aag atc atc aat ggg ggc gag ctc ttt ctt act gtg ctc ctc aat 2009 Asp Lys Ile Ile Asn Gly Glu Leu Phe Leu Thr Val Leu Leu Asn 510 515 520 cct atc agc gtc ttc atg aca cac tta tcc aac tat gga aat gac cgc 2057 Pro Ile Ser Val Phe Met Thr His Leu Ser Asn Tyr Gly Asn Asp Arg 525 530 530 535 ctg ggact tac acc ttc aag cac ctg gtg cgc ttc ctg cac tcc tgg 2105 Leu Gly Leu Tyr Thr Phe Lys His Leu Val Arg Phe Leu His Ser Trp 540 545 550 550 acc aac ctg agg ctg cag acg ctg ccc cct gtg cag ag gcc 153 Thr Asn Leu Arg Leu Gln Thr Leu Pro Pro Val Gln Leu Ala Gln Lys 555 560 565 tac ttc cag atc ttt tct gag gag aag gac cca ctt tgg cag gat ccc 2201 Tyr Phe Gln Ile Phe Ser Glu Glu Lys Asp Pro Leu Trp Gln Asp Pro 570 575 580 585 tgt gag gac aaa cgc cac aaa gac atc tgg tct aag gag aag aca tgt 2249 Cys Glu Asp Lys Arg His Lys Asp Ile Trp Ser Lys Glu Lys Thr Cys 590 595 600 gat cgc ttc cca aag ctg c tc atc att ggc ccc cag aaa aca ggc acc 2297 Asp Arg Phe Pro Lys Leu Leu Ile Ile Gly Pro Gln Lys Thr Gly Thr 605 610 615 aca gcc ctc tac ctg ttc ctg ggc atg cac ccc gac ctc agc Agc agc agc Agc Agc Agc Agc Agc Agc Agc Agc Tyr Leu Phe Leu Gly Met His Pro Asp Leu Ser Ser Asn 620 625 630 tac ccc agc tcc gag acc ttt gag gag atc cag ttt ttt aat ggc cac 2393 Tyr Pro Ser Ser Glu Thr Phe Glu Glu Ile Gln Phe Phe Asn Gly His 635 640 645 aac tat cac aaa ggc atc gac tgg tac atg gaa ttc ttc cct att ccc 2441 Asn Tyr His Lys Gly Ile Asp Trp Tyr Met Glu Phe Phe Pro Ile Pro 650 655 660 665 tcc aac acc acc tct gac ttc tac ttt ga agt gcc aac tac ttt 2489 Ser Asn Thr Thr Ser Asp Phe Tyr Phe Glu Lys Ser Ala Asn Tyr Phe 670 675 680 gat tca gaa gtg gca cca cgg cga gca gct gcc cta ttg ccc aag gcc 2537 Asp Ser Glu Val Ala Pro Arg Arg Ala Ala Ala Leu Leu Pro Lys Ala 685 690 695 aag gtt ctc acc atc ctc atc aat cca gcc gac cgg gct tac tcc tgg 2585 Lys Val Leu Thr Ile Leu Ile Asn Pro Ala Asp Arg Ala Tyr Ser Trp 700 705 710 tac ca g cac cag cgg gcc cat gat gac ccg gtg gcc cta aag tac acc 2633 Tyr Gln His Gln Arg Ala His Asp Asp Pro Val Ala Leu Lys Tyr Thr 715 720 725 ttc cat gag gtg atc aca gct ggc cct gac gca tcc tca aag ctg cgt 2681 Phe His Glu Val Ile Thr Ala Gly Pro Asp Ala Ser Ser Lys Leu Arg 730 735 740 745 gcc ctc cag aac cga tgc ctg gtc ccc ggc tgg tat gcc act cat att 2729 Ala Leu Gln Asn Arg Cys Leu Val Pro Gly Trp Tyr Ala Thr His Ile 750 755 760 gaa cgc tgg ctc agc gcc ttt cat gcc aac cag atc ctg gtc ttg gat 2777 Glu Arg Trp Leu Ser Ala Phe His Ala Asn Gln Ile Leu Val Leu Asp 765 770 775 ggc c aaa ctg gaa cct gcc aaa gtg atg gac aca gtg cag 2825 Gly Lys Leu Leu Arg Thr Glu Pro Ala Lys Val Met Asp Thr Val Gln 780 785 790 aaa ttc ctc ggg gtg acc agc acg gtt gac tac cat aaa acc ttg gcg 2873 Lys Gly Val Thr Ser Thr Val Asp Tyr His Lys Thr Leu Ala 795 800 805 ttt gac cca aag aaa gga ttt tgg tgc cag ctg ctc gaa gga gga aaa 2921 Phe Asp Pro Lys Lys Gly Phe Trp Cys Gln Leu Leu Glu Gly Gly Lys 810 815 820 825 acc aag tgt ctg gga aaa agc aag gga cgg aaa tat cca gag atg gac 2969 Thr Lys Cys Leu Gly Lys Ser Lys Gly Arg Lys Tyr Pro Glu Met Asp 830 835 835 ctg gat tcc cga gcc ttc cta agat tac cgg gac cac aac att 3017 Leu Asp Ser Arg Ala Phe Leu Lys Asp Tyr Tyr Arg Asp His Asn Ile 845 850 855 gag ctc tct aag ctg ctg tat aag atg ggc cag aca ctg ccc acc tgg 3065 Glu Leu Tyr Leu Lys Met Gly Gln Thr Leu Pro Thr Trp 860 865 870 ctg cgg gaa gac ctc cag aac acc agg tag ccttggccac cacagccagc 3115 Leu Arg Glu Asp Leu Gln Asn Thr Arg 875 880 cagaacgctt gtgttagcag ggatgtcctg cctcacactg agccagactg acctgcctcg 3175 aaggatgctg gccccagcca gccaggagca acgagcaata ccctgctaag gcccaccaga 3235 gccggaagcc caggcaggtc tgcaagcgcc tcagagcatc cactgctgga tgtgtggctg 3295 tgggacctct gtgggaccag aggtccattc cgttccttcg cagcctccct gcctggggag 3355 agcacttcct gttggtggaa ggtcatttcc tggtaggagg agtcctggag actctctcct 3415 gtccctcact gtgttcggcc agtcctgccc tgttctgtgt cataccaccc ctgctccagc 3475 aggatgtccc ctcagtatta gctgtcatat ttctctgtcc tccagacagt aagggagagg 3535 agcgcagctg ggcctctcgc ccaactagag agaaagactg ggcatgtccc tgagggtttg 3595 agccaggccc cgccagggtt taggtaggca cccagatgca ctcatagatt gaatgtgagg 3655 gtggccatct tgagaggaca tacgactcag tatttgggtt attagtatca atctcatctc 3715 ccctttgggg gaaagactct ctggtcccta ttgtatccac ctagtgctca tggtctcttg 3775 ttggccctgg gccactgccc tgccactggg cccagagaca tgggccttgg ccctgtcctg 3835 ttcacctgga tgtgacctgt ggtgtttcct gtggtaaagg ctgaggcgag tcaggagtct 3895 gccagtgttc atactcccat gtacatatac actgtctccc agccaccgcc tcggcccggc 3955 aggcaagcag agtcagcagc actgctctct actgctttgc ctggcaacct gtggctgagg 4015 gtccccagag acccccccaa cctcccaaat actaag 4051 <210> 2 <211> 882 <212> PRT <213> Rattus sp. <400> 18 Met Pro Ala Leu Ala Cys Leu Arg Arg Leu Cys Arg His Leu Ser Pro 1 5 10 15 Gln Ala Val Leu Phe Leu Leu Phe Val Phe Cys Leu Phe Ser Val Phe 20 25 30 Val Ser Ala Tyr Tyr Leu Tyr Gly Trp Asn Arg Gly Leu Glu Pro Ser 35 40 45 Ala Asp Ala Ser Glu Ser Asp Cys Gly Asp Pro Pro Pro Val Ala Pro 50 55 60 Ser Arg Leu Leu Pro Ile Lys Pro Val Gln Ala Val Ala Pro Ser Arg 65 70 75 80 Thr Asp Pro Leu Val Leu Val Phe Val Glu Ser Leu Tyr Ser Gln Leu 85 90 95 Gly Gln Glu Val Val Ala Ile Leu Glu Ser Ser Arg Phe Lys Tyr Arg 100 105 110 Thr Glu Ile Ala Pro Gly Lys Gly Asp Met Pro Thr Leu Thr Asp Lys 115 120 125 Gly Arg Gly Arg Phe Ala Leu Ile Ile Tyr Glu Asn Ile Leu Lys Tyr 130 135 140 Val Asn Leu Asp Ala Trp Asn Arg Glu Leu Leu Asp Lys Tyr Cys Val 145 150 155 160 Ala Tyr Gly Val Gly Ile Ile Gly Phe Phe Lys Ala Asn Glu Asn Ser 165 170 175 Leu Leu Ser Ala Gln Leu Lys Gly Phe Pro Leu Phe Leu His Ser Asn 180 185 190 Leu Gly Leu Lys Asp Cys Ser Ile Asn Pro Lys Ser Pro Leu Leu Tyr 195 200 205 Val Thr Arg P ro Ser Glu Val Glu Lys Gly Val Leu Pro Gly Glu Asp 210 215 220 Trp Thr Val Phe Gln Ser Asn His Ser Thr Tyr Glu Pro Val Leu Leu 225 230 235 240 Ala Lys Thr Arg Ser Ser Glu Ser Ile Pro His Leu Gly Ala Asp Ala 245 250 255 Gly Leu His Ala Ala Leu His Ala Thr Val Val Gln Asp Leu Gly Leu 260 265 270 His Asp Gly Ile Gln Arg Val Leu Phe Gly Asn Asn Leu Asn Phe Trp 275 280 285 285 Leu His Lys Leu Val Phe Val Asp Ala Val Ala Phe Leu Thr Gly Lys 290 295 300 Arg Leu Ser Leu Pro Leu Asp Arg Tyr Ile Leu Val Asp Ile Asp Asp 305 310 315 320 Ile Phe Val Gly Lys Glu Gly Thr Arg Met Lys Val Glu Asp Val Lys 325 330 335 Ala Leu Phe Asp Thr Gln Asn Glu Leu Arg Thr His Ile Pro Asn Phe 340 345 350 Thr Phe Asn Leu Gly Tyr Ser Gly Lys Phe Phe His Thr Gly Thr Asp 355 360 365 Ala Glu Asp Ala Gly Asp Asp Leu Leu Leu Ser Tyr Val Lys Glu Phe 370 375 380 Trp Trp Phe Pro His Met Trp Ser His Met Gln Pro His Leu Phe His 385 390 395 400 400 Asn Gln Ser Val Leu Ala Glu Gln Met Ala Leu Asn Lys Lys Phe Ala 405 410 415 Val Glu His G ly Ile Pro Thr Asp Met Gly Tyr Ala Val Ala Pro His 420 425 430 His Ser Gly Val Tyr Pro Val His Val Gln Leu Tyr Glu Ala Trp Lys 435 440 445 Gln Val Trp Asn Ile Arg Val Thr Ser Thr Glu Glu Tyr Pro His Leu 450 455 460 Lys Pro Ala Arg Tyr Arg Arg Gly Phe Ile His Asn Gly Ile Met Val 465 470 475 480 Leu Pro Arg Gln Thr Cys Gly Leu Phe Thr His His Thr Ile Phe Tyr Asn 485 490 490 495 Glu Tyr Pro Gly Gly Ser Ser Glu Leu Asp Lys Ile Ile Asn Gly Gly 500 505 510 Glu Leu Phe Leu Thr Val Leu Leu Asn Pro Ile Ser Val Phe Met Thr 515 520 525 His Leu Ser Asn Tyr Gly Asn Asp Arg Leu Gly Leu Tyr Thr Phe Lys 530 535 540 His Leu Val Arg Phe Leu His Ser Trp Thr Asn Leu Arg Leu Gln Thr 545 550 555 560 Leu Pro Pro Val Gln Leu Ala Gln Lys Tyr Phe Gln Ile Phe Ser Glu 565 570 575 Glu Lys Asp Pro Leu Trp Gln Asp Pro Cys Glu Asp Lys Arg His Lys 580 585 590 Asp Ile Trp Ser Lys Glu Lys Thr Cys Asp Arg Phe Pro Lys Leu Leu 595 600 600 605 Ile Ile Gly Pro Gln Lys Thr Gly Thr Ala Leu Tyr Leu Phe Leu 610 615 620 Gly Met His Pro A sp Leu Ser Ser Asn Tyr Pro Ser Ser Glu Thr Phe 625 630 635 640 Glu Glu Ile Gln Phe Phe Asn Gly His Asn Tyr His Lys Gly Ile Asp 645 650 655 Trp Tyr Met Glu Phe Phe Pro Ile Pro Ser Asn Thr Thr Ser Serp Phe 660 665 670 Tyr Phe Glu Lys Ser Ala Asn Tyr Phe Asp Ser Glu Val Ala Pro Arg 675 680 685 Arg Ala Ala Ala Leu Leu Pro Lys Ala Lys Val Leu Thr Ile Leu Ile 690 695 700 Asn Pro Ala Asp Arg Ala Tyr Ser Trp Tyr Gln His Gln Arg Ala His 705 710 715 715 720 Asp Asp Pro Val Ala Leu Lys Tyr Thr Phe His Glu Val Ile Thr Ala 725 730 735 Gly Pro Asp Ala Ser Ser Lys Leu Arg Ala Leu Gln Asn Arg Cys Leu 740 745 750 Val Pro Gly Trp Tyr Ala Thr His Ile Glu Arg Trp Leu Ser Ala Phe 755 760 765 His Ala Asn Gln Ile Leu Val Leu Asp Gly Lys Leu Leu Arg Thr Glu 770 775 780 Pro Ala Lys Val Met Asp Thr Val Gln Lys Phe Leu Gly Val Thr Ser 785 790 795 800 Thr Val Asp Tyr His Lys Thr Leu Ala Phe Asp Pro Lys Lys Gly Phe 805 810 815 Trp Cys Gln Leu Leu Glu Gly Gly Lys Thr Lys Cys Leu Gly Lys Ser 820 825 830 Lys Gly Arg Lys T yr Pro Glu Met Asp Leu Asp Ser Arg Ala Phe Leu 835 840 845 Lys Asp Tyr Tyr Arg Asp His Asn Ile Glu Leu Ser Lys Leu Leu Tyr 850 855 860 Lys Met Gly Gln Thr Leu Pro Thr Trp Leu Arg Glu Asp Leu Gln Asn 865 870 875 880 Thr Arg <210> 3 <211> 1551 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA segment from Rat N-heparan sulfate sulfotransferase cDNA <400> 3 tggaaccggg gcctcgagcc ctcggcagat gcttctgagt ccgactgcgg ggacccacca 60 cctgtcgccc ctagccgtct cctgccaatc aagcctgtgc aggcggtcgc cccttctcga 120 acagacccgc tggtgctggt atttgtggag agcctctatt cacagctggg ccaggaggtg 180 gtggccatcc tggaatccag tcgcttcaag taccgaacag aaattgcacc ggggaagggg 240 gacatgccca cactcacaga caagggccga ggccgcttcg ccctcatcat ctatgagaac 300 atcctcaagt atgtcaacct ggatgcctgg aaccgggagc tgctggacaa gtactgtgtg 360 gcctacggcg tgggcatcat tggcttcttc aaggccaatg agaacagcct gctgagtgca 420 cagctcaaag gcttccctct tttcctgcat tcgaacctgg gcttgaaaga ctgcagcatc 480 aaccccaagt ccccactgct gtacgtgaca cggcccagtg aggtagagaa aggtgtgctg 540 cccggagagg actggacggt gttccagtct aaccactcta cctatgagcc agtgctgctg 600 gccaagacgc gctcctctga gtccatccca cacctgggcg cagatgccgg cctgcatgct 660 gccctgcacg ctactgtggt ccaggacctg ggcctccatg acggcattca gcgtgtgctg 720 tttggcaaca acctcaactt ttggctgcat aagctcgtct tcgtggacgc tgtggccttc 780 ctcacaggga agcgcctctc actgcctttg gaccgataca tcctggtgga cattgatgac 840 atttttgtag gc aaggaggg cacacgcatg aaggtggagg atgtgaaggc cctgtttgat 900 acacagaatg aacttcgtac acatatccca aacttcacct tcaacctggg ctactcaggg 960 aaattcttcc acacaggtac cgatgctgag gatgctgggg acgacctgct gctgtcctat 1020 gtgaaagagt tctggtggtt cccccacatg tggagccata tgcaacccca cctcttccac 1080 aaccagtctg tgctggctga gcagatggcc ctgaacaaga agttcgctgt cgagcacggc 1140 attcccacag atatggggta tgcagtggca ccccaccact ctggtgtgta ccctgtgcat 1200 gtgcagctgt atgaggcctg gaagcaagtg tggaacatcc gtgtgaccag cacagaggag 1260 tacccgcatc tgaagcctgc ccgttaccgc cgtggcttca tccacaatgg catcatggtc 1320 ctccctcggc agacctgtgg tctctttaca cacaccatct tctacaacga gtaccctgga 1380 ggctccagtg agctggacaa gatcatcaat gggggcgagc tctttcttac tgtgctcctc 1440 aatcctatca gcgtcttcat gacacactta tccaactatg gaaatgaccg cctgggactg 1500 tacaccttca agcacctggt gcgcttcctg cactcctgga ccaacctgag g 1551 <210> 4 <211> 3901 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (217) .. (2865) <400> 4 gaaggaagga gcgtgaccag cctgtggact gcgcccctgg ctgggaggaa ggactggggg 60 cccagatcct ccactcccag tgccccacaa gggcgtcgct tcctaagtct ctgtgaattt 120 gttggtcagt ggacgattct cgtgtctcct cctgtgtggg gccttggggt agccagggca 180 ggccgggcct ccgtggccaa ggtctcggag gccagg atg cct gcc ctg gca tgc 234 Met Pro Ala Leu Ala Cys 1 5 ctc cgg agg ctg tgt cgg cac gtg tcc ccg cag gct gtc ctt ttc ctg 282 Leu Arg Arg Leu Cys Arg His Val Ser Pro Gln Ala Val Leu Phe Leu 10 15 20 ctg ttc atc ttc tgc ctg ttc agc gtt ttc atc tcg gcc Lec tac Tta cta gta Phe Cys Leu Phe Ser Val Phe Ile Ser Ala Tyr Tyr Leu 25 30 35 tat ggc tgg aag cga ggc ctg gag ccc tcg gcg gat gcc ccc gag cct 378 Tyr Gly Trp Lys Arg Gly Leu Glu Pro Ser Ala Asp Ala Pro Glu Pro 40 45 50 gac tgc ggg gac ccg ccg cct gtg gcc ccc agt cgc ctg ctg cca ctc 426 Asp Cys Gly Asp Pro Pro Pro Val Ala Pro Ser Arg Leu Leu Pro Leu 55 60 65 70 aag cct gtg cag gca gcc acc cct tcc cgc aca gac ccg ttg gtg ctg 474 Lys Pro Val Gln Ala Ala Thr Pro Ser Arg Thr Asp Pro Leu Val Leu 75 80 85 gtc ttt gtg gag agc ctc tac tcg caa ctg ggc cag gag gtg gtg gcc 522 Val Phe Val Glu Ser Leu Tyr Ser Gln Leu Gly Gln Glu Val Val Ala 90 95 100 atc ctg gag tcc agc cgc ttc tac cgc aca gag att gcg ccg ggc 570 Ile Leu Glu Ser Ser Arg Phe Lys Tyr Arg Thr Glu Ile Ala Pro Gly 105 110 115 aag ggt gac atg ccc acg ctc act gac aag ggc cgt ggc cgc ttc gcc Met 618 Lys Gly Asp Thr Leu Thr Asp Lys Gly Arg Gly Arg Phe Ala 120 125 130 ctc atc atc tat gag aac atc ctc aag tat gtc aac ctg gac gcc tgg 666 Leu Ile Ile Tyr Glu Asn Ile Leu Lys Tyr Val Asn Leu Asp Ala Trp 135 140 145 150 aac cgg gag ctg ctg gac aag tac tgt gtg gcc tac ggc gtg ggc atc 714 Asn Arg Glu Leu Leu Asp Lys Tyr Cys Val Ala Tyr Gly Val Gly Ile 155 160 165 att ggc ttc ttc aag gcc aatgag ag ag gag ag gag ag gcg cag ctc 762 Ile Gly Phe Phe Lys Ala Asn Glu Asn Ser Leu Leu Ser Ala Gln Leu 170 175 180 aag ggc ttc ccc ctg ttc ctg cac tca aac ctg ggc ctg aag gac tgc 810 Lys Gly Phe Pro Leu Phe Pro Leu Phe Pro Leu Leu G ly Leu Lys Asp Cys 185 190 195 agc atc aac ccc aag tcc ccg ctg ctc tac gtg acg cga cct agc gag 858 Ser Ile Asn Pro Lys Ser Pro Leu Leu Tyr Val Thr Arg Pro Ser Glu 200 205 210 gtg gag aaa ggt gtg ctc ccc ggc gag gac tgg acg gtt ttc cag tca 906 Val Glu Lys Gly Val Leu Pro Gly Glu Asp Trp Thr Val Phe Gln Ser 215 220 225 230 aat cac tcc acc tat gag cca gtg ctg ctg gcc aag acg cgc tcg tct 954 Hisn Ser Thr Tyr Glu Pro Val Leu Leu Ala Lys Thr Arg Ser Ser 235 240 245 gag tcc atc cca cac ctg ggc gca gac gcc ggc ctg cat gct gca ctg 1002 Glu Ser Ile Pro His Leu Gly Ala Asp Ala Gly Leu His Ala Ala Leu 250 255 260 cac gcc act gtg gtc cag gac ctg ggc ctg cac gac ggc atc cag cgc 1050 His Ala Thr Val Val Gln Asp Leu Gly Leu His Asp Gly Ile Gln Arg 265 270 275 275 gtg ctg ttt ggc aac aac ctg act ttg cac aag ctt gtc ttc 1098 Val Leu Phe Gly Asn Asn Leu Asn Phe Trp Leu His Lys Leu Val Phe 280 285 290 gtg gat gcc gtg gcc ttc ctc acg ggg aag cgc ctc tcc ctg cca ttg 1146 Val Asp Ala Ala Val hr Gly Lys Arg Leu Ser Leu Pro Leu 295 300 305 310 gac cgc tac atc ctg gtg gac att gat gac atc ttc gtg ggc aag gag 1194 Asp Arg Tyr Ile Leu Val Asp Ile Asp Asp Ile Phe Val Gly Lys Glu 315 320 325 aca cgc atg aag gtg gag gac gtg aag gcc ctg ttt gac aca cag 1242 Gly Thr Arg Met Lys Val Glu Asp Val Lys Ala Leu Phe Asp Thr Gln 330 335 340 aac gaa cta cgc gca cac atc cca aac ttc acc ttc ac ttc acc ttc ag tac 1290 Asn Glu Leu Arg Ala His Ile Pro Asn Phe Thr Phe Asn Leu Gly Tyr 345 350 355 tca ggg aaa ttc ttc cac aca ggt acc aat gct gag gac gct ggg gat 1338 Ser Gly Lys Phe Phe His Thr Gly Thr Asn Ala Glu Asp Ala Gly Asp 360 365 370 gat ctg ctg ctg tcg tat gtg aag gag ttc tgg tgg ttc ccc cac atg 1386 Asp Leu Leu Leu Ser Tyr Val Lys Glu Phe Trp Trp Phe Pro His Met 375 380 385 385 390 tgg agccc at cac ctt ttc cac aac cag tcc gtg ttg gcc 1434 Trp Ser His Met Gln Pro His Leu Phe His Asn Gln Ser Val Leu Ala 395 400 405 gag cag atg gcc ttg aac aag aag ttc gct gtc gag cat ggc att ccc 1482 Glu Gl n Met Ala Leu Asn Lys Lys Phe Ala Val Glu His Gly Ile Pro 410 415 420 aca gac atg ggg tat gca gtg gcg ccc cac cac tcg ggc gtg tac ccc 1530 Thr Asp Met Gly Tyr Ala Val Ala Pro His His Ser Gly Val Tyr Pro 425 430 435 gtg cac gtg cag ctg tac gag gct tgg aag cag gtg tgg agc atc cgc 1578 Val His Val Gln Leu Tyr Glu Ala Trp Lys Gln Val Trp Ser Ile Arg 440 445 450 gtg acc agc acg gag gag tac ccc c aag cca gcc cgc tac cgc 1626 Val Thr Ser Thr Glu Glu Tyr Pro His Leu Lys Pro Ala Arg Tyr Arg 455 460 465 470 cgt ggc ttc atc cac aat ggc atc atg gtt ctc cca cgg cag acc tgc 1674 Arg Gly Phe Ile Asn Gly Ile Met Val Leu Pro Arg Gln Thr Cys 475 480 485 ggc ctc ttc aca cac acc atc ttc tac aac gag tac cct ggc ggc tcc 1722 Gly Leu Phe Thr His Thr Ile Phe Tyr Asn Glu Tyr Pro Gly Gly Ser 490 495 495 500 agt gag ctg gac aaa atc atc aac ggg ggc gag ctc ttc ctc acc gtg 1770 Ser Glu Leu Asp Lys Ile Ile Asn Gly Gly Glu Leu Phe Leu Thr Val 505 510 515 ctc ctc aat cct atc agc atc ttc atg acg acc tat ggg 1818 Leu Leu Asn Pro Ile Ser Ile Phe Met Thr His Leu Ser Asn Tyr Gly 520 525 530 aat gac cgc ctg ggc ctg tac acc ttc aag cac ctg gtg cgc ttc ctg 1866 Asn Asp Arg Leu Gly Leu Tyr Thr Leu Val Arg Phe Leu 535 540 545 550 cac tcc tgg acg aac ctc cgg ctg cag aca ctg ccc cct gtg cag ttg 1914 His Ser Trp Thr Asn Leu Arg Leu Gln Thr Leu Pro Pro Val Gln Leu 555 560 565 565 gcg cag aag tac ttg cag atc ttc tcc gag gag aag gac ccg ctc tgg 1962 Ala Gln Lys Tyr Phe Gln Ile Phe Ser Glu Glu Lys Asp Pro Leu Trp 570 575 580 cag gac ccc tgc gag gac aaa cgt cac aaa gac atc tgg tcc aag gag 2010 Pro Cys Glu Asp Lys Arg His Lys Asp Ile Trp Ser Lys Glu 585 590 595 aag acg tgt gac cgc ttc cca aag ctc ctc atc atc ggc ccc cag aaa 2058 Lys Thr Cys Asp Arg Phe Pro Lys Leu Leu Ile Ile Gly Pro Gln 600 605 610 aca ggc acc act gcc ctc tac ctg ttc ctg ggc atg cac cct gac cta 2106 Thr Gly Thr Thr Ala Leu Tyr Leu Phe Leu Gly Met His Pro Asp Leu 615 620 625 630 agc agc aac tac ccc agc tct gag cc ttt gag gag atc cag ttt ttt 2154 Ser Ser Asn Tyr Pro Ser Ser Glu Thr Phe Glu Glu Ile Gln Phe Phe 635 640 645 aat ggc cac aac tat cac aaa ggc atc gac tgg tac atg gag ttc ttc 2202 Asn Gly His Asn Tyr His Lys Gly Ile Asp Trp Tyr Met Glu Phe Phe Phe 650 655 660 ccc atc cct tcc aac acc acc tcc gac ttc tac ttt gag aaa agc gcc 2250 Pro Ile Pro Ser Asn Thr Thr Ser Asp Phe Tyr Phe Glu Lys Ser Ala 665 670 675 aac tac ttt gat tca gaa gtg gcg ccc cgg cgg gca gca gcc ctc ttg 2298 Asn Tyr Phe Asp Ser Glu Val Ala Pro Arg Arg Ala Ala Ala Ala Leu Leu 680 685 690 ccc aaa gcc aag gtc ctg acc atc ccc atc ag ac cgg gcc 2346 Pro Lys Ala Lys Val Leu Thr Ile Leu Ile Asn Pro Ala Asp Arg Ala 695 700 705 710 tat tcc tgg tac cag cac cag cga gcc cat gac gac cca gtg gcc cta 2394 Tyr Ser Trp Tyr Gln His Gln Arg Ala His Asp Asp Pro Val Ala Leu 715 720 725 aag tac acc ttc cat gag gtg att acc gcc ggc tct gac gca tcc tcg 2442 Lys Tyr Thr Phe His Glu Val Ile Thr Ala Gly Ser Asp Ala Ser Ser 730 735 740 aag ctg cgt gc c ctc cag aac cgc tgc ctg gtc cct ggc tgg tac gcc 2490 Lys Leu Arg Ala Leu Gln Asn Arg Cys Leu Val Pro Gly Trp Tyr Ala 745 750 755 acc cac atc gag cgc tgg ctc agt gcc tat cac atcc gac cag 538 Thr His Ile Glu Arg Trp Leu Ser Ala Tyr His Ala Asn Gln Ile Leu 760 765 770 gtc ttg gat ggc aaa ctg ctt cgc aca gaa cct gcc aaa gtg atg gac 2586 Val Leu Asp Gly Lys Leu Leu Arg Thr Glu Pro Ala Met Asp 775 780 785 790 atg gtg cag aag ttc ctt ggg gtg acc aac acc att gac tac cac aaa 2634 Met Val Gln Lys Phe Leu Gly Val Thr Asn Thr Ile Asp Tyr His Lys 795 800 805 acc ttg gcg ttt gat cca aag aaa gga ttt tgg tgc caa ctg ctt gaa 2682 Thr Leu Ala Phe Asp Pro Lys Lys Gly Phe Trp Cys Gln Leu Leu Glu 810 815 820 gga gga aaa acc aag tgt ctg ggc aaa agc aag ggc cgg aaa tat ccc 2730 Lysly Gly Cys Leu Gly Lys Ser Lys Gly Arg Lys Tyr Pro 825 830 835 gag atg gac ttg gat tcc cga gcc ttc ctg aag gac tat tac cgg gac 2778 Glu Met Asp Leu Asp Ser Arg Ala Phe Leu Lys Asp Tyr Tyr Arg Asp 840 845 850 cac aac atc gag ctc tcc aag ctg ctg tat aag atg ggc cag aca ctt 2826 His Asn Ile Glu Leu Ser Lys Leu Leu Tyr Lys Met Gly Gln Thr Leu 855 860 865 870 870 ccc act tgg cta cga gag gac ctc cag aac tag ccgtggccac 2875 Pro Thr Trp Leu Arg Glu Asp Leu Gln Asn Thr Arg 875 880 cacagccaga ctgaacgttt gtgaaagctg ggacatccca ccacacgctg agccagacct 2935 gcagagtggg aagctggacc agggcagctg cgcacttatg agcaatactc tgtggaggtc 2995 tggtggggct gggggagcac ccaggcggat ctgcaagcac ctcggagcac ccaccgctgg 3055 gtctgcggcc taagggacct ccctcgccag cagaggtcca ttccgttccc agctgctcct 3115 ggggaggccg cttcctggta ggagggagtc cacgagactc ttttctgtcc ctcactgtgt 3175 tccgccgact gtcccctctc gtcacccatc actccctgct tccgcagggc gcccctcagt 3235 attcgctgcc atatgtccct gtcctccagg ctgtagggga ggagagcctg gccgggggag 3295 acagactgga catttccctg tttcgagcca ggctcttcca aggggccagc tgggtccccg 3355 gagtcagtcc taggctggat gggagggtgg ccccctcaag aggactccca gcctccacat 3415 ctggttccta ccttcacatc tcaccctccc gttctgggga agaatttctg gttcctacag 3475 tatccactcc a tcctcaagg cttcccgcag ggccttgggg cactgccttg ccatcgggcc 3535 cagttctccg ggccccacct gcaccccttt cttccccctg ggatatgatg tgtggtgttt 3595 cctgtggtaa aagactgagg caggccaggg gtctgccagt aacatgttcc catgtacaga 3655 cacggtcccc acaccctccc agcctcaggc ccaggcagac atgggcgagc tggtgagact 3715 gccagccacg gctttgctta gccacctgtg gccgagggct ctcagagacc cccttaacct 3775 cccaaatact aagaagctaa aatattttaa tattttgttt ttttttttct tggtgccaga 3835 gtttataccc tgggtgctgg ggtcgcactg tgttatatat atatatatat atatatataa 3895 tgtgta 3901 <210> 5 <211> 882 <212> PRT <213> Homo sapiens <400> 19 Met Pro Ala Leu Ala Cys Leu Arg Arg Leu Cys Arg His Val Ser Pro 1 5 10 15 Gln Ala Val Leu Phe Leu Leu Phe Ile Phe Cys Leu Phe Ser Val Phe 20 25 30 Ile Ser Ala Tyr Tyr Leu Tyr Gly Trp Lys Arg Gly Leu Glu Pro Ser 35 40 45 Ala Asp Ala Pro Glu Pro Asp Cys Gly Asp Pro Pro Pro Val Ala Pro 50 55 60 Ser Arg Leu Leu Pro Leu Lys Pro Val Gln Ala Ala Thr Pro Ser Arg 65 70 75 80 Thr Asp Pro Leu Val Leu Val Phe Val Glu Ser Leu Tyr Ser Gln Leu 85 90 95 Gly Gln Glu Val Val Ala Ile Leu Glu Ser Ser Arg Phe Lys Tyr Arg 100 105 110 Thr Glu Ile Ala Pro Gly Lys Gly Asp Met Pro Thr Leu Thr Asp Lys 115 120 125 Gly Arg Gly Arg Phe Ala Leu Ile Ile Tyr Glu Asn Ile Leu Lys Tyr 130 135 140 Val Asn Leu Asp Ala Trp Asn Arg Glu Leu Leu Asp Lys Tyr Cys Val 145 150 155 160 Ala Tyr Gly Val Gly Ile Ile Gly Phe Phe Lys Ala Asn Glu Asn Ser 165 170 175 Leu Leu Ser Ala Gln Leu Lys Gly Phe Pro Leu Phe Leu His Ser Asn 180 185 190 Leu Gly Leu Lys Asp Cys Ser Ile Asn Pro Lys Ser Pro Leu Leu Tyr 195 200 205 Val Thr Arg P ro Ser Glu Val Glu Lys Gly Val Leu Pro Gly Glu Asp 210 215 220 Trp Thr Val Phe Gln Ser Asn His Ser Thr Tyr Glu Pro Val Leu Leu 225 230 235 240 Ala Lys Thr Arg Ser Ser Glu Ser Ile Pro His Leu Gly Ala Asp Ala 245 250 255 Gly Leu His Ala Ala Leu His Ala Thr Val Val Gln Asp Leu Gly Leu 260 265 270 His Asp Gly Ile Gln Arg Val Leu Phe Gly Asn Asn Leu Asn Phe Trp 275 280 285 285 Leu His Lys Leu Val Phe Val Asp Ala Val Ala Phe Leu Thr Gly Lys 290 295 300 Arg Leu Ser Leu Pro Leu Asp Arg Tyr Ile Leu Val Asp Ile Asp Asp 305 310 315 320 Ile Phe Val Gly Lys Glu Gly Thr Arg Met Lys Val Glu Asp Val Lys 325 330 335 Ala Leu Phe Asp Thr Gln Asn Glu Leu Arg Ala His Ile Pro Asn Phe 340 345 350 Thr Phe Asn Leu Gly Tyr Ser Gly Lys Phe Phe His Thr Gly Thr Asn 355 360 365 Ala Glu Asp Ala Gly Asp Asp Leu Leu Leu Ser Tyr Val Lys Glu Phe 370 375 380 Trp Trp Phe Pro His Met Trp Ser His Met Gln Pro His Leu Phe His 385 390 395 400 400 Asn Gln Ser Val Leu Ala Glu Gln Met Ala Leu Asn Lys Lys Phe Ala 405 410 415 Val Glu His G ly Ile Pro Thr Asp Met Gly Tyr Ala Val Ala Pro His 420 425 430 His Ser Gly Val Tyr Pro Val His Val Gln Leu Tyr Glu Ala Trp Lys 435 440 445 Gln Val Trp Ser Ile Arg Val Thr Ser Thr Glu Glu Tyr Pro His Leu 450 455 460 Lys Pro Ala Arg Tyr Arg Arg Gly Phe Ile His Asn Gly Ile Met Val 465 470 475 480 Leu Pro Arg Gln Thr Cys Gly Leu Phe Thr His His Thr Ile Phe Tyr Asn 485 490 490 495 Glu Tyr Pro Gly Gly Ser Ser Glu Leu Asp Lys Ile Ile Asn Gly Gly 500 505 510 Glu Leu Phe Leu Thr Val Leu Leu Asn Pro Ile Ser Ile Phe Met Thr 515 520 525 His Leu Ser Asn Tyr Gly Asn Asp Arg Leu Gly Leu Tyr Thr Phe Lys 530 535 540 His Leu Val Arg Phe Leu His Ser Trp Thr Asn Leu Arg Leu Gln Thr 545 550 555 560 Leu Pro Pro Val Gln Leu Ala Gln Lys Tyr Phe Gln Ile Phe Ser Glu 565 570 575 Glu Lys Asp Pro Leu Trp Gln Asp Pro Cys Glu Asp Lys Arg His Lys 580 585 590 Asp Ile Trp Ser Lys Glu Lys Thr Cys Asp Arg Phe Pro Lys Leu Leu 595 600 600 605 Ile Ile Gly Pro Gln Lys Thr Gly Thr Ala Leu Tyr Leu Phe Leu 610 615 620 Gly Met His Pro A sp Leu Ser Ser Asn Tyr Pro Ser Ser Glu Thr Phe 625 630 635 640 Glu Glu Ile Gln Phe Phe Asn Gly His Asn Tyr His Lys Gly Ile Asp 645 650 655 Trp Tyr Met Glu Phe Phe Pro Ile Pro Ser Asn Thr Thr Ser Serp Phe 660 665 670 Tyr Phe Glu Lys Ser Ala Asn Tyr Phe Asp Ser Glu Val Ala Pro Arg 675 680 685 Arg Ala Ala Ala Leu Leu Pro Lys Ala Lys Val Leu Thr Ile Leu Ile 690 695 700 Asn Pro Ala Asp Arg Ala Tyr Ser Trp Tyr Gln His Gln Arg Ala His 705 710 715 715 720 Asp Asp Pro Val Ala Leu Lys Tyr Thr Phe His Glu Val Ile Thr Ala 725 730 735 Gly Ser Asp Ala Ser Ser Lys Leu Arg Ala Leu Gln Asn Arg Cys Leu 740 745 750 Val Pro Gly Trp Tyr Ala Thr His Ile Glu Arg Trp Leu Ser Ala Tyr 755 760 765 His Ala Asn Gln Ile Leu Val Leu Asp Gly Lys Leu Leu Arg Thr Glu 770 775 780 Pro Ala Lys Val Met Asp Met Val Gln Lys Phe Leu Gly Val Thr Asn 785 790 795 800 Thr Ile Asp Tyr His Lys Thr Leu Ala Phe Asp Pro Lys Lys Gly Phe 805 810 815 Trp Cys Gln Leu Leu Glu Glu Gly Gly Lys Thr Lys Cys Leu Gly Lys Ser 820 825 830 Lys Gly Arg Lys T yr Pro Glu Met Asp Leu Asp Ser Arg Ala Phe Leu 835 840 845 Lys Asp Tyr Tyr Arg Asp His Asn Ile Glu Leu Ser Lys Leu Leu Tyr 850 855 860 Lys Met Gly Gln Thr Leu Pro Thr Trp Leu Arg Glu Asp Leu Gln Asn 865 870 875 880 Thr Arg <210> 6 <211> 1551 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: DNA segment from Human N-heparan sulfate sulfotransferase cDNA <400> 6 tggaagcgag gcctggagcc ctcggcggat gcccccgagc ctgactgcgg ggacccgccg 60 cctgtggccc ccagtcgcct gctgccactc aagcctgtgc aggcagccac cccttcccgc 120 acagacccgt tggtgctggt ctttgtggag agcctctact cgcaactggg ccaggaggtg 180 gtggccatcc tggagtccag ccgcttcaaa taccgcacag agattgcgcc gggcaagggt 240 gacatgccca cgctcactga caagggccgt ggccgcttcg ccctcatcat ctatgagaac 300 atcctcaagt atgtcaacct ggacgcctgg aaccgggagc tgctggacaa gtactgtgtg 360 gcctacggcg tgggcatcat tggcttcttc aaggccaatg agaacagcct gctgagtgcg 420 cagctcaagg gcttccccct gttcctgcac tcaaacctgg gcctgaagga ctgcagcatc 480 aaccccaagt ccccgctgct ctacgtgacg cgacctagcg aggtggagaa aggtgtgctc 540 cccggcgagg actggacggt tttccagtca aatcactcca cctatgagcc agtgctgctg 600 gccaagacgc gctcgtctga gtccatccca cacctgggcg cagacgccgg cctgcatgct 660 gcactgcacg ccactgtggt ccaggacctg ggcctgcacg acggcatcca gcgcgtgctg 720 tttggcaaca acctgaactt ctggctgcac aagcttgtct tcgtggatgc cgtggccttc 780 ctcacgggga agcgcctctc cctgccattg gaccgctaca tcctggtgga cattgatgac 840 atcttcgtgg gc aaggaggg cacacgcatg aaggtggagg acgtgaaggc cctgtttgac 900 acacagaacg aactacgcgc acacatccca aacttcacct tcaacctggg ctactcaggg 960 aaattcttcc acacaggtac caatgctgag gacgctgggg atgatctgct gctgtcgtat 1020 gtgaaggagt tctggtggtt cccccacatg tggagccaca tgcagcccca ccttttccac 1080 aaccagtccg tgttggccga gcagatggcc ttgaacaaga agttcgctgt cgagcatggc 1140 attcccacag acatggggta tgcagtggcg ccccaccact cgggcgtgta ccccgtgcac 1200 gtgcagctgt acgaggcttg gaagcaggtg tggagcatcc gcgtgaccag cacggaggag 1260 tacccccacc tgaagccagc ccgctaccgc cgtggcttca tccacaatgg catcatggtt 1320 ctcccacggc agacctgcgg cctcttcaca cacaccatct tctacaacga gtaccctggc 1380 ggctccagtg agctggacaa aatcatcaac gggggcgagc tcttcctcac cgtgctcctc 1440 aatcctatca gcatcttcat gacgcacctg tccaactatg ggaatgaccg cctgggcctg 1500 tacaccttca agcacctggt gcgcttcctg cactcctgga cgaacctccg g 1551 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 7 atataaagct tatgcctgcc ctggcgtgcc tc 32 <210> 8 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 8 atataaagct ttggaaccgg ggcctcgagc cc 32 <210> 9 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 9 atataaagct tgtcctccct cggcagacct gt 32 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 10 atataaagct tctgcagacg ctgccccctg tg 32 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 11 gcgcaagctt actacctggt gttctggagg tc 32 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 12 gcgcaagctt acctcaggtt ggtccaggag tg 32 <210> 13 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 13 gcgcaagctt acatgatgcc attgtggatg aa 32 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 14 atataaagct tatgcctgcc ctggcatgcc tc 32 <210> 15 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 15 atataaagct ttggaagcga ggcctggagc cc 32 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 16 atataaagct tgttctccca cggcagacct gc 32 <210> 17 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forword) <400> 17 atataaagct tctgcagaca ctgccccctg tg 32 <210> 18 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 18 gcgcaagctt accggaggtt cgtccaggag tg 32 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sewuence: primer for RT-PCR (reverse) <400> 19 gcgcaagcca tgatgccatt gtggatgaag cc 32 <210> 20 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR (forword) <400> 20 taaaccatgg ggggttctca tcatcatcat cat 33 <210> 21 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for PCR (reverse) <400> 21 atatccatgg atacctcctt ccaagcttac ctcaggttgg 40

[Brief description of the drawings]

FIG. 1 shows hydropathy plots of human and rat NDST.

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 4B024 BA10 CA04 CA07 DA06 EA03 EA04 GA11 HA01 HA03 4B050 CC01 CC03 DD11 FF14E LL10 4B065 AA26X AA90Y AB01 BA02 BD14 CA29 CA60

Claims (9)

[Claims] 1. A DNA encoding a partial polypeptide of an N-acetylglucosamine N-deacetylated / N-sulfotransferase polypeptide, which is expressed by inserting the DNA into an expression vector. The N-acetylglucosamine N-deacetylated N-sulfotransferase is such that the obtained partial polypeptide can have an action of deacetylating the N-acetylglucosamine residue of the sugar chain having a heparin skeleton. DNA which does not have the amino terminal region and the carboxyl terminal region of the polypeptide. 2. DNA of 2 kbp or less capable of hybridizing under stringent conditions to a DNA having the nucleotide sequence of SEQ ID NO: 3 or 6 or a nucleotide sequence complementary thereto.
A DNA encoding a polypeptide having an action of deacetylating an N-acetylglucosamine residue of a sugar chain having a heparin skeleton. 3. An expression vector containing the DNA according to claim 1 or 2. 4. The expression vector according to claim 3, further comprising a DNA encoding a polypeptide having an activity of transferring a sulfate group to an amino group of a glucosamine residue. 5. A transformant obtained by transforming a host cell with the expression vector according to claim 3 or 4. 6. The transformant according to claim 5, wherein the host cell is a microorganism. 7. The transformant according to claim 6, wherein the microorganism is Escherichia coli. 8. An enzyme obtainable by growing the transformant according to any one of claims 5 to 7, wherein the enzyme deacetylates an N-acetylglucosamine residue of a sugar chain having a heparin skeleton. N-acetylglucosamine deacetylase lacking a transmembrane region having an action. 9. A composition comprising the transmembrane region-deficient N-acetylglucosamine deacetylase according to claim 8 and an N-sulfotransferase.