JP4224581B2 - Thermostable enzyme having sugar nucleotide synthesis activity and DNA encoding the enzyme - Google Patents

Description

【００１４】
また、この反応においては。上記精製した酵素のみならず、粗酵素であってもよい。例えば、宿主として枯草菌等分泌型の系を用いる場合には、培養液中に本酵素が生成蓄積され、大腸菌等の非分泌型の系を用いる場合には、菌体内に生成されるので、本酵素を含有する培養液あるいはその処理物、もしくは菌体破砕物等の培養処理物を用いて、糖ヌクレオチドを合成してもよい。
以下に、本発明の実施例を示すが、本発明実施例により限定されるものではない。
【００１５】
【実施例１】
糖ヌクレオチド合成酵素の製造
（１）菌の培養
好酸性好気性超好熱古細菌スルフォロバス、トーコーダイイJCM10545は次の方法で培養した。
1.3ｇの(NH₄)₂SO₄、0.28gのKH₂PO₄、0.25gのMgSO₄・7H₂O、0.07gのCaCl₂・2H₂O、0.02gのFeCl₃・6H₂O、1.8mg のMnCl₂・4H₂O、4.5mgのNa₂B₄O₇・10H₂O、0.22mgのZnSO₄・7H₂O、0.05mgのCuCl₂・2H₂O、0.03mgのNa₂MoO₄・2H₂O、0.03mgのVOSO₄・xH₂O、0.01mgのCoSO₄・7H₂O、1.0gの酵母エキスを1Lの蒸留水に溶かし、この溶液のpHを3.5に10規定H₂SO₄溶液で調製した。加圧殺菌した後、JCM10545を植菌した。この培養液を80℃で1〜2日培養し、その後遠心分離し集菌した。
【００１６】
（2）染色体ＤＮＡの調製
JCM10545の染色体DNAは以下の方法により調製した。
培養終了後5000rpm、10分間の遠心分離により菌体を集菌する。菌体を10 mM EDTA(pH 6.0)溶液で洗浄後、50 mM Tris/HCl-50 mM EDTA (pH8.5)溶液を加えて細胞を溶解させる。さらに、0.5% Na-lauroylsarcosinate、1 mg/ml プロテアーゼKとなるように各々を加えた後、50℃で3時間保温する。フェノール処理を３回行った後、溶液を10 mM Tris-10 mM EDTA (pH 8.0)溶液に対して透析する。37℃で30分間のRNaseによるRNAの分解後、フェノールクロロフォルム溶液で処理した後、10 mM Tris-1 mM EDTA(pH 8.0)で透析を行う。
【００１７】
（3）染色体DNAを含むショットガンライブラリークローンの作製
実施例2で得られた染色体DNAを超音波処理することにより断片化した後、アガロースゲル電気泳動により1kb及び2kb長のDNA断片を回収した。この断片をプラスミドベクターpUC118のHincII制限酵素部位に挿入したショットガンライブラリーを作製した。各ショットガンクローンの末端塩基配列を、ABI社製自動塩基配列読み取り装置377を用いて解読していった。各ショットガンクローンから得られた塩基配列を塩基配列自動連結ソフトSequencherを用いて連結編集し、本菌の全塩基配列を決定していった。
【００１８】
（4）STRmlA1-3遺伝子の同定
上記手法で決定された好酸性好気性超好熱古細菌スルフォロバス、トーコーダイイのゲノム塩基配列の大型計算機による解析を行い、グルコース1リン酸チミジル酸結合酵素の機能を含むであろうタンパク質をコードする遺伝子（ST0452, ST1971, ST2352）を３個同定した。この３個の内、超好熱性古細菌スルフォロバストーコーダイイのST0452遺伝子の開始コドンはATGで、401アミノ酸残基のタンパク質をコードする遺伝子として同定された。
【００１９】
（5）発現プラスミドの構築
構造遺伝子領域の前後に制限酵素（NdeIとXhoI）サイトを構築する目的でDNAプライマーを合成し、PCRでその遺伝子の前後に制限酵素サイトを導入した。その際に合成されるタンパク質のC末端にヒスチジン残基をタグとして結合するように合成されるようにする場合とST0452遺伝子がコードするタンパク質のみを合成させるようにする場合とでプライマーの配列が異なる。
【００２０】
Upper primer,
5'- ATAGCATATGAAGGCATTTATTCTTGCTGC -3'（配列番号１）（下線部はNdeIサイトを示す）
Lower prime 1, ヒスチジン残基を結合させる場合
5'- TCAACTCGAGGACCTTGAAAAACTCACC-3'（配列番号２）（下線部はXhoIサイトを示す）
Lower prime 2, ヒスチジン残基を結合させ無い場合
5'- TCAACTCGAGCTAGACCTTGAAAAACTCACC -3'（配列番号３）（下線部はXhoIサイトを示す）
【００２１】
Upper primerとLower primer1或いはLower primer2を組み合わせたPCR反応後、制限酵素（NdeIとXhoI）で完全分解（37℃で2時間）した後、その構造遺伝子領域断片を精製した。
制限酵素NdeIとXhoIで切断後精製したpET21b（Novagen社製）と上記の構造遺伝子(ST0452)領域断片とをT4リガーゼを用いて16℃、２時間反応させることによって連結した。連結したDNAの一部を大腸菌DH5αのコンピテントセルに導入し形質転換体のコロニーを得た。得られたコロニーからプラスミドをQIAprep Spin Miniprep Kit（QIAGEN社製）で精製し、塩基配列を確認して発現プラスミド、pET21b/ST0452-1及びpET21b/ST0452-2を得た。発現プラスミドpET21b/ST0452-1を用いるとSTRmlA1はC末端にヒスチジンタグが付加された融合タンパク質として生産され、発現プラスミドpET21b/ST0452-2を用いるとSTRmlA1はC末端にヒスチジンタグが付加されないタンパク質として生産される。
【００２２】
（6）組換え遺伝子の発現
大腸菌（E. coli BL21(DE3) CodonPlus RIL,、Novagen社製）のコンピテントセルを融解して、二本のファルコンチューブに各々0.1mlづつ移す。その中に上記の２種の発現プラスミド10ng分に相当する溶液を別々に加え氷中に30分間放置した後42℃でヒートショックを30秒間行い、そこにSOC培地0.9mlを加え、37℃で1時間振とう培養する。その後、アンピシリンを含むLB寒天プレート上に適量まき、37℃で一晩培養し、形質転換体大腸菌 BL21(DE3) CodonPlus RIL/pET21b/ST0452-1及び形質転換体大腸菌 BL21(DE3) CodonPlus RIL/pET21b/ST0452-2を得た。
【００２３】
当該形質転換体をアンピシリンを含むLB培地（２リットル）中で一晩37℃において培養した後、IPTG（Isopropyl-b-D-thiogalactopyranoside）を1mMになるように加え、さらに30℃で5時間培養した。培養後遠心分離（6,000ｒｐｍ，20min）により集菌を行った。
【００２４】
（7）STRmlA1酵素の精製
8リットル培養液から集菌した菌体に２倍量の40mMトリス塩酸緩衝液（pH8.0）、1錠のプロテアーゼ阻害剤（Complete EDTA-free, Roche社製）、0.5mgのDnase RQ1（プロメガ社製）を加え懸濁液を得た。得られた懸濁液を超音波破砕し、75℃で10分保温した後、遠心分離(11,000 rpm、20分)により上清液を得た。この上清液を用いNi-カラム（Novagen, His・Bind metal chelation resin & His・Bind buffer kitを使用）による親和性クロマトグラムを行った。ここで得られた0.5 Mイミダゾール溶出画分（20ml）を、再度75℃で10分加熱処理し、遠心分離(11,000 rpm、20分)により上清液を得た。次にこの上清液を20mMトリス塩酸緩衝液(pH8.0)、2.5M NaClで平衡化したHiTrap phenyl sepharose（ファルマシア社製）カラムに吸着させ、同緩衝液中のNaCl濃度を2.5Mから1Mに低下させることにより、目的タンパク質の溶出を行った。さらに、セントリプレップYM-50 (アミコン社)で2mlまで濃縮し、これを20mMトリス塩酸緩衝液（pH8.0）、100mM NaClで透析し、精製サンプルとした。
【００２５】
【実施例２】
糖ヌクレオチドの合成
（１）糖ヌクレオチド合成反応（糖とヌクレオチド結合反応）
50mM Tris緩衝液（pH7.5）、12 mM MgCl₂、24 mM Glucose-1-phosphate、1 mM TTP、1 Uのinorganic pyrophosphataseからなる酵素反応液300μl中に実施例１で得られた精製酵素0.0135 mgを加えた。この酵素反応液を80℃で保温することにより、反応させた。5分10分15分20分25分後に30μl を分取し、300 μl の500 mM KH₂PO₄溶液に加える事により反応停止させた。
【００２６】
(2) 糖ヌクレオチド合成反応（糖とヌクレオチドの結合反応）の測定
HPLCを用いて、反応生成物であるTDP-Glucoseの量を、ヌクレオチド部分の紫外線の吸収を目安に測定した。図１に示すように標準物質であるTTP及びTDP-Glucoseは、HPLCにおいて溶出位置が全く異なる。さらに、図２に示すように標準サンプル添加量を変化させたときの、ピークの面積と標準物質量は正確な比例関係にあり、この検量線を用いることにより反応生成物を定量出来る事が示された。
そこで、上記(1)で反応させたサンプルに関しても、HPLCで同様の解析を行った。
【００２７】
【実施例３】
酵素の性質
（1）タンパク質化学的性質
当該酵素は上記の精製プロセスで完全に精製され、SDS-PAGEで分子量約44KDaの単一バンドを示した（図３）。当該酵素は401アミノ酸残基より構成され（配列番号４）、そのアミノ酸配列から予測される分子量は44,000 Daであった。また、結核菌RmlAとの相同性は低いが、ヌクレオチド認識に関与するモチーフが保存されていた（図４）。
【００２８】
(２) 糖ヌクレオチド合成活性（糖とヌクレオチドの結合活性）
当該酵素は37℃においては、図５に有るように糖ヌクレオチド合成活性をほとんど示さないが、80℃においては高い酵素活性を示した。活性の発現には一度、80℃20分間の加熱処理が必須であるが、加熱後であっても37℃での活性は非常に低い（図６）。
【００２９】
(３) 熱安定性
50mM Tris緩衝液（pH7.5）、12 mM MgCl₂、24 mM Glucose-1-phosphate、1 mM TTP、1 Uのinorganic pyrophosphataseからなる酵素反応液300μl中に、あらかじめ80℃で5分10分20分30分40分60分90分120分間加熱した実施例１で得られた精製酵素0.0135 mgを加えた。この酵素反応液を80℃で5分間保温することにより、反応させた後に30μl を分取し、300 μl の500 mM KH₂PO₄溶液に加える事により反応停止させた。反応の進行は、実施例２の(2)に有るようにHPLCで測定した。その結果、図７に示すように、本酵素は80℃による90分間の加熱処理後でも、50％以上の活性を残すことから非常に安定で耐熱性が高いことが示された。
【００３０】
（４）pH依存性
12 mM MgCl₂、24 mM Glucose-1-phosphate、1 mM TTP、1 Uのinorganic pyrophosphataseからなる酵素反応液300μl中の50mM Tris緩衝液のpHを（pH 2）、（pH 4）、（pH 6）、（pH 6.5）、（pH 7）、（pH 7.5）、（pH 8）、（pH 10）に変化させた酵素反応液中に、実施例１で得られた精製酵素0.0135 mgを加えた。この酵素反応液を80℃で5分間保温することにより、反応させた後に30μl を分取し、300 μl の500 mM KH₂PO₄溶液に加える事により反応停止させた。反応の進行は、実施例２の(2)に有るようにHPLCで測定した。
図８に示すように、本酵素の活性はpH7.5において最も高い活性を示した。
【００３１】
（５）ヌクレオシド３リン酸基質の多様性
50mM Tris緩衝液（pH7.5）、12 mM MgCl₂、24 mM Glucose-1-phosphate、1 Uのinorganic pyrophosphatase及び1 mM dTTPまたは1 mM dATP、1 mM dGTP、1 mM dCTP、1 mM UTP、1 mM ATP、1 mM GTP、1 mM CTPからなる酵素反応液300μl中に実施例１で得られた精製酵素0.0135 mgを加えた。この酵素反応液を80℃で5分間保温することにより、反応させた後に30μl を分取し、300 μl の500 mM KH₂PO₄溶液に加える事により反応停止させた。反応の進行は、実施例２の(2)に有るようにHPLCで測定した。結果を表1に示す。表１から明らかなように、本酵素はdTTP以外にdATP、dGTP、dCTP、UTPを基質として利用出来ることが示された。
【００３２】
【表１】
各種ヌクレオシド３リン酸の基質としての利用性

【００３３】
（６）糖１リン酸基質の多様性
50mM Tris緩衝液（pH7.5）、12 mM MgCl₂、1 mM TTP、1 Uのinorganic pyrophosphatase及び24 mM α-D-Glucose-1-phosphateまたは24 mM D-fructose-1-phosphate、24 mM α-D-mannose-1-phosphate、24 mM α-D-Galactose-1-phosphateからなる酵素反応液300μl中に実施例１で得られた精製酵素0.0135 mgを加えた。この酵素反応液を80℃で5分間保温することにより、反応させた後に30μl を分取し、300 μl の500 mM KH₂PO₄溶液に加える事により反応停止させた。反応の進行は、実施例２の(2)に有るようにHPLCで測定した。表２に示すように、本酵素はα-D-Glucose-1-phosphate以外にD-fructose-1-phosphate及びα-D-mannose-1-phosphateを基質として利用出来ることが示された。
【００３４】
【表２】
各糖一リン酸の基質としての利用性

【００３５】
【発明の効果】
本発明により、試験管内での様々な種類の糖ヌクレオチドを合成することが可能で、かつ熱等に安定な新規な糖ヌクレオチド合成酵素が提供できた。その結果、新規な糖ヌクレオチド合成が可能になった。
一方、糖ヌクレオチドは、糖タンパク質、糖脂質、多糖類の糖鎖合成に糖供与体として機能するものであり、これらの糖鎖合成は、癌転移、器官発生あるいは細胞性免疫等に密接に関連するものとして近年注目されており、本発明は、これら研究の発展において、その貢献度は極めて大きい。
【００３６】
【配列表】

【図面の簡単な説明】
【図１】 ＨＰＬＣによる、TTP、及びTTPとTDP-Glucose混合物の分離パターンを測定したグラフである。
【図２】 ＨＰＬＣを用いたTDP-Glucoseの検量線を示す図である。
【図３】 精製されたSTRmlA1蛋白質のSDS-PAGEパターンを示す写真である。
【図４】 結核菌（M.tuberculosis)の.RmlA蛋白質と本発明の酵素遺伝子（ST0452,ST1971,ST2352)によりコードされる各蛋白質のアミノ酸配列、及びこれらのタンパク質間で保存された配列モチーフを示す図である。
（なお、数字は、蛋白質アミノ基末端側からの残基数を示す。）
【図５】 STRmlA1蛋白質の37℃における糖ヌクレオチド合成活性を示すグラフである。
【図６】 STRmlA1蛋白質の80℃における糖ヌクレオチド合成活性を示すグラフである。
【図７】 STRmlA1蛋白質の80℃処理における残存活性量を示す図である。
【図８】 STRmlA1蛋白質のｐＨの違いによる活性変化を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention uses a heat-resistant protein having sugar nucleotide synthesis activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant having the recombinant DNA, and the transformant The present invention relates to a method for producing a protein having sugar nucleotide synthesis activity, and a method for producing a sugar nucleotide using the protein or the transformant.
[0002]
[Prior art]
As an enzyme having a sugar nucleotide (TDP-Glucose) synthesis activity, Mycobacterium tuberculosis (see Non-patent document 1), Pseudomonas aeruginosa (see Non-patent document 2), Salmonella enterica (non-patent document) Detailed properties of RmlA (Glucose-1-phosphate thymidylyltransferase) derived from Reference 3) have already been reported. RmlA is initial enzyme essential metabolic pathway rhamnose synthesis, glucose-1-phosphate and nucleobase shea de triphosphate as substrate, it produces a nucleobase shea de triphosphate glucose. In addition, enzymes that synthesize sugar nucleotides from humans and yeasts have been found, but most of them are derived from cold organisms, so they are extremely unstable at room temperature and above, and their activity is quickly lost by heat treatment at about 80 ° C. Live. For this reason, treatments such as sterilization at the time of use are necessary, and careful storage at low temperatures is necessary.
[0003]
[Non-Patent Document 1]
Yufang Ma, Jonathan A. Mills, John T. Belisle, Vara Vissa, Mark Howell, Kelly Bowlin, Michael S. Scherman and Michael McNeil "Determination of the pathway for rhamnose biosynthesis in mycobacteria: cloning, sequencing and expression of the Mycobacterium tuberculosis gene encoding α-D-glucose-1-phosphate thymidylyltransferase “(1997) Microbiology, 143, 937-945.
[Non-Patent Document 2]
Wulf Blankenfeldt, Miryam Asuncion, Joseph S. Lam and James H. Naismith “The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA)” (2000) EMBO J., 19, 6652-6663.
[Non-Patent Document 3]
Lennart Lindquist, Rudorf Kaiser, Peter R. Reeves and Alf A. Lindberg "Purification, characterization and HPLC assay of Salmonella glucose-1-phosphate thymidylyltransferase from the cloned rfbA gene" (1993) Eur. J. Biochem., 211, 763- 770.
[0004]
[Problems to be solved by the invention]
If a thermostable enzyme having an activity of synthesizing sugar nucleotides is discovered, it becomes possible to stably synthesize sugar nucleotides serving as a substrate for sugar chain synthesis. In addition, it is expensive to find an enzyme that can catalyze a binding reaction using various types of sugars necessary as a substrate for sugar chain synthesis as well as various types of nucleoside triphosphates such as UTP / GTP as substrates. This makes it possible to synthesize many kinds of unstable sugar nucleotides. Furthermore, stable enzymes that perform these reactions have been craved.
[0005]
Accordingly, an object of the present invention is to provide a novel enzyme having heat resistance and capable of synthesizing sugar nucleotides using various sugars and nucleoside triphosphates as substrates.
[0006]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventor focused on the hyperthermophilic archaeon Sulfolobus tokodaii strain7 that grows at 75-80 ° C., and the gene presumed to have this enzyme activity from the whole genome gene information. Searched for. Furthermore, an enzyme is produced from the gene using Escherichia coli, and it is confirmed that this enzyme is stably present at high temperature (80 ° C.) and exhibits sugar nucleotide synthesis activity. It has been found that various kinds of sugar nucleotides can be produced, and the present invention has been completed.
[0007]
That is, the present invention is as follows.
(1) Except when the following sugar monophosphate consisting of (a) and nucleoside triphosphate consisting of (b) are used as substrates (however, glucose-1-phosphate and TTP (thymidine triphosphate ) are used as substrates ) As
(A) Glucose-1-phosphate, mannose-1-phosphate, or fructose-1-phosphate (b) TTP (thymidine triphosphate), dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate) Fate), dCTP (deoxycytidine triphosphate), or UTP (uridine triphosphate).
A protein having the amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 4; A method for producing a sugar nucleotide, which comprises allowing a protein having the protein to act . "
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Below, this invention is demonstrated concretely.
The hyperthermophilic archaea used in the present invention is an acidophilic aerobic hyperthermophilic archaeon sulforobes, Toko Daii (JCM registration number JCM10545). Three gene regions estimated to show this enzyme activity from the whole genome information of this hyperthermophilic archaea were amplified and extracted by PCR reaction, inserted into the protein expression plasmid pET21b, and then transformed with that plasmid. This enzyme was produced. The produced enzyme was isolated and purified by heat treatment and column chromatogram. Of the purified enzymes, STRmlA1 was found to be a protein having a molecular weight of about 44,000 and an activity to synthesize various sugar nucleotides.
[0009]
The half-life of this enzyme was high in heat resistance at 50 ° C. for 40 minutes or more in 50 m Tris-HCl buffer (pH 7.5).
The amino acid sequence of this STRmlA1 and the base sequence of its gene DNA (ST0452) are shown in SEQ ID NOs: 4 and 7, respectively.
The amino acid sequences of the enzymes (STRmlA2 and STRmlA3) encoded by the remaining two gene regions are shown in SEQ ID NOs: 5 and 6, respectively, and the base sequences of these gene DNAs (ST1971 and ST2352) are shown in the same SEQ ID NOs. 8 and 9.
[0010]
The enzyme in the present invention is not limited to those having the amino acid sequences shown in SEQ ID NOs: 4 to 6, and in the amino acid sequence, one or more amino acid residues are deleted, substituted, inserted or added. Even so, proteins having this amino acid sequence include those that exhibit sugar nucleotide synthesis activity. In addition, these enzyme gene DNAs of the present invention are not limited to those having the base sequences shown in SEQ ID NOs: 7 to 9, but include those encoding the amino acid sequences. Furthermore, a DNA that hybridizes to the DNA represented by any of SEQ ID NOs: 7 to 9 under a stringent condition and encodes a protein having sugar nucleotide synthesis activity is also included. These stringent conditions include 52.59 g NaCl, 26.46 g sodium citrate, 1 g Ficoll (Type 400), 1 g polyvinylpyrrolidone, 1 g bovine serum albumin, 5 g SDS, 1 g in 1 liter of hybridization solution. It contains fragmented sperm DNA, 500 ml formamide, and is performed at a temperature of 42 ° C. Subsequent washings consisted of 17.53 g NaCl, 8.82 g sodium citrate, 5 liters in 1 liter of washing solution.
gContains SDS at a temperature of 68 ° C.
[0011]
In order to obtain the enzyme of the present invention, ordinary genetic engineering techniques can be applied. The above-mentioned various enzyme gene DNAs are inserted into, for example, protein expression plasmid vectors such as pET21b and pHY481, and a recombinant vector is prepared. A host cell is transformed with the vector, the transformant is cultured in a medium, and the enzyme is isolated from the culture, culture-treated product, or transformant separated and recovered from the culture by a conventional protein purification means. Purify and isolate. As the host cell, Escherichia coli, Bacillus subtilis and the like can be used.
[0012]
In the present invention, this enzyme is further used to synthesize a sugar nucleotide. In this synthesis, the enzyme is added to a solution containing sugar monophosphate and nucleoside triphosphate, and the reaction temperature is from 60 ° C. to React at 95 ° C to obtain sugar nucleotides.
Examples of sugar monophosphate include sugar-1-phosphate such as glucose-1-phosphate, mannose-1-phosphate, and fructose-1-phosphate. Examples of nucleoside triphosphate include TTP (thymidine triphosphate), dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dCTP (deoxycytidine triphosphate), GTP (guanosine triphosphate), UTP (uridine triphosphate) Etc.
[0013]
As a reaction formula, a case where glucose nucleotides are synthesized from glucose-1-phosphate and TTP is shown below.
[Chemical 1]

[0014]
Also in this reaction. Not only the purified enzyme but also a crude enzyme may be used. For example, when a secretory system such as Bacillus subtilis is used as a host, the enzyme is produced and accumulated in the culture solution, and when a non-secretory system such as Escherichia coli is used, it is produced in the bacterial body. Sugar nucleotides may be synthesized using a culture solution containing the present enzyme, a processed product thereof, or a cultured product such as a crushed bacterial cell.
Examples of the present invention will be shown below, but the present invention is not limited to the examples.
[0015]
[Example 1]
Production of sugar nucleotide synthase (1) Culture of bacteria Acidophilic aerobic super thermophilic archaea Sulfolobus, Tokodai JCM10545 was cultured by the following method.
1.3 g (NH ₄ ) ₂ SO ₄ , 0.28 g KH ₂ PO ₄ , 0.25 g MgSO ₄ .7H ₂ O, 0.07 g CaCl ₂ .2H ₂ O, 0.02 g FeCl ₃ .6H ₂ O, 1.8 mg MnCl ₂ 4H ₂ O, 4.5 mg Na ₂ B ₄ O ₇ · 10H ₂ O, 0.22 mg ZnSO ₄ · 7H ₂ O, 0.05 mg CuCl ₂ · 2H ₂ O, 0.03 mg Na ₂ MoO ₄・ 2H ₂ O, 0.03 mg VOSO ₄・ xH ₂ O, 0.01 mg CoSO ₄・ 7H ₂ O, 1.0 g yeast extract was dissolved in 1 L distilled water, and the pH of this solution was adjusted to 3.5 with 10 N H ₂ SO Prepared with ₄ solutions. After sterilization under pressure, JCM10545 was inoculated. This culture solution was cultured at 80 ° C. for 1-2 days, and then centrifuged to collect bacteria.
[0016]
(2) Preparation of chromosomal DNA
The chromosomal DNA of JCM10545 was prepared by the following method.
After culturing, the cells are collected by centrifugation at 5000 rpm for 10 minutes. After washing the cells with 10 mM EDTA (pH 6.0) solution, 50 mM Tris / HCl-50 mM EDTA (pH 8.5) solution is added to lyse the cells. Furthermore, after adding each so that it may become 0.5% Na-lauroylsarcosinate and 1 mg / ml protease K, it heat-retains at 50 degreeC for 3 hours. After three phenol treatments, the solution is dialyzed against 10 mM Tris-10 mM EDTA (pH 8.0) solution. After degradation of RNA with RNase at 37 ° C. for 30 minutes, treatment with phenol chloroform solution is followed by dialysis against 10 mM Tris-1 mM EDTA (pH 8.0).
[0017]
(3) Production of shotgun library clones containing chromosomal DNA Fragmented chromosomal DNA obtained in Example 2 was subjected to sonication, and 1 kb and 2 kb long DNA fragments were recovered by agarose gel electrophoresis. . A shotgun library was prepared by inserting this fragment into the HincII restriction enzyme site of plasmid vector pUC118. The terminal base sequence of each shotgun clone was decoded using an automatic base sequence reader 377 manufactured by ABI. The base sequence obtained from each shotgun clone was ligated and edited using the base sequence automatic linking software Sequencher, and the entire base sequence of this bacterium was determined.
[0018]
(4) Identification of the STRmlA1-3 gene A large computer analysis of the genomic base sequences of the eosinophilic, aerobic, hyperthermophilic archaeon Sulfolobus and Tokodaii determined by the above method was performed to determine the function of glucose 1-phosphate thymidylate-binding enzyme. Three genes (ST0452, ST1971, ST2352) encoding proteins that would be included were identified. Of these three, the start codon of the ST0452 gene of the hyperthermophilic archaeon Sulfolobastocodaii was ATG, which was identified as a gene encoding a protein of 401 amino acid residues.
[0019]
(5) Construction of expression plasmid DNA primers were synthesized for the purpose of constructing restriction enzyme (NdeI and XhoI) sites before and after the gene region, and restriction enzyme sites were introduced before and after the gene by PCR. The primer sequence differs depending on whether the protein is synthesized so that the histidine residue is bound as a tag to the C-terminus of the protein synthesized or when only the protein encoded by the ST0452 gene is synthesized. .
[0020]
Upper primer,
5′-ATAG CATATG AAGGCATTTATTCTTGCTGC -3 ′ (SEQ ID NO: 1) (underlined indicates NdeI site)
Lower prime 1, for binding histidine residues
5'-TCAA CTCGAG GACCTTGAAAAACTCACC-3 '(SEQ ID NO: 2) (underlined indicates XhoI site)
Lower prime 2, when histidine residue is not bound
5'-TCAA CTCGAG CTAGACCTTGAAAAACTCACC-3 '(SEQ ID NO: 3) (underlined indicates XhoI site)
[0021]
After a PCR reaction combining Upper primer and Lower primer 1 or Lower primer 2, it was completely digested with restriction enzymes (NdeI and XhoI) (2 hours at 37 ° C), and then the structural gene region fragment was purified.
PET21b (manufactured by Novagen) purified by digestion with restriction enzymes NdeI and XhoI and the above structural gene (ST0452) region fragment were ligated by reacting at 16 ° C. for 2 hours using T4 ligase. A part of the ligated DNA was introduced into competent cells of E. coli DH5α to obtain transformant colonies. Plasmids were purified from the obtained colonies using QIAprep Spin Miniprep Kit (manufactured by QIAGEN), and the nucleotide sequences were confirmed to obtain expression plasmids, pET21b / ST0452-1 and pET21b / ST0452-2. Using expression plasmid pET21b / ST0452-1, STRmlA1 is produced as a fusion protein with a histidine tag added to the C-terminus, and using expression plasmid pET21b / ST0452-2, STRmlA1 is produced as a protein without a histidine tag added to the C-terminus Is done.
[0022]
(6) Recombinant gene expression Thaw competent cells of E. coli BL21 (DE3) CodonPlus RIL, manufactured by Novagen, and transfer 0.1 ml each to two falcon tubes. The solution corresponding to 10 ng of the above-mentioned two expression plasmids was added separately, and left on ice for 30 minutes, followed by heat shock at 42 ° C. for 30 seconds, and then 0.9 ml of SOC medium was added thereto at 37 ° C. Incubate with shaking for 1 hour. Thereafter, an appropriate amount is sprinkled on an LB agar plate containing ampicillin and cultured at 37 ° C. overnight. / ST0452-2 was obtained.
[0023]
The transformant was cultured overnight at 37 ° C. in LB medium (2 liters) containing ampicillin, and then IPTG (Isopropyl-bD-thiogalactopyranoside) was added to 1 mM and further cultured at 30 ° C. for 5 hours. Bacteria were collected after centrifugation by centrifugation (6,000 rpm, 20 min).
[0024]
(7) Purification of STRmlA1 enzyme
Cells collected from 8 liter culture solution are doubled in 40 mM Tris-HCl buffer (pH 8.0), 1 tablet protease inhibitor (Complete EDTA-free, manufactured by Roche), 0.5 mg Dnase RQ1 (Promega) To obtain a suspension. The obtained suspension was sonicated and incubated at 75 ° C. for 10 minutes, and then a supernatant was obtained by centrifugation (11,000 rpm, 20 minutes). The supernatant was used for affinity chromatography using a Ni-column (using Novagen, His • Bind metal chelation resin & His • Bind buffer kit). The 0.5 M imidazole elution fraction (20 ml) obtained here was again heat-treated at 75 ° C. for 10 minutes, and a supernatant was obtained by centrifugation (11,000 rpm, 20 minutes). Next, this supernatant was adsorbed to a 20 mM Tris-HCl buffer (pH 8.0) and HiTrap phenyl sepharose (Pharmacia) column equilibrated with 2.5 M NaCl, and the NaCl concentration in the buffer was adjusted from 2.5 M to 1 M. The protein of interest was eluted by lowering to a low level. Further, the solution was concentrated to 2 ml with Centriprep YM-50 (Amicon) and dialyzed with 20 mM Tris-HCl buffer (pH 8.0) and 100 mM NaCl to obtain a purified sample.
[0025]
[Example 2]
Synthesis of sugar nucleotides (1) Sugar nucleotide synthesis reaction (sugar and nucleotide binding reaction)
The purified enzyme 0.0135 obtained in Example 1 in 300 μl of an enzyme reaction solution consisting of 50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, 1 U of inorganic pyrophosphatase mg was added. The enzyme reaction solution was kept at 80 ° C. for reaction. After 5 minutes, 10 minutes, 15 minutes, 20 minutes and 25 minutes, 30 μl was collected and added to 300 μl of 500 mM KH ₂ PO ₄ solution to stop the reaction.
[0026]
(2) Measurement of sugar nucleotide synthesis reaction (sugar-nucleotide binding reaction)
Using HPLC, the amount of TDP-Glucose, which is a reaction product, was measured based on the absorption of ultraviolet light at the nucleotide moiety. As shown in FIG. 1, TTP and TDP-Glucose, which are standard substances, have completely different elution positions in HPLC. Furthermore, as shown in FIG. 2, the peak area and the amount of the standard substance when the amount of the standard sample added is changed are in an accurate proportional relationship, and it is shown that the reaction product can be quantified by using this calibration curve. It was done.
Therefore, the same analysis was performed on the sample reacted in (1) above by HPLC.
[0027]
[Example 3]
Properties of enzyme (1) Protein chemistry The enzyme was completely purified by the above purification process and showed a single band with a molecular weight of about 44 KDa on SDS-PAGE (FIG. 3). The enzyme was composed of 401 amino acid residues (SEQ ID NO: 4), and the molecular weight predicted from the amino acid sequence was 44,000 Da. Moreover, although the homology with Mycobacterium tuberculosis RmlA was low, the motif involved in nucleotide recognition was preserve | saved (FIG. 4).
[0028]
(2) Sugar nucleotide synthesis activity (sugar-nucleotide binding activity)
The enzyme showed almost no sugar nucleotide synthesis activity at 37 ° C. as shown in FIG. 5, but showed high enzyme activity at 80 ° C. A heat treatment at 80 ° C. for 20 minutes is essential once for the expression of the activity, but the activity at 37 ° C. is very low even after heating (FIG. 6).
[0029]
(3) Thermal stability
In an enzyme reaction solution consisting of 50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, 1 U of inorganic pyrophosphatase in advance at 80 ° C. for 5 minutes 10 minutes 20 0.0135 mg of the purified enzyme obtained in Example 1 heated for 30 minutes, 40 minutes, 60 minutes, 90 minutes and 120 minutes was added. The enzyme reaction solution was kept at 80 ° C. for 5 minutes to react, and 30 μl was collected and added to 300 μl of 500 mM KH ₂ PO ₄ solution to stop the reaction. The progress of the reaction was measured by HPLC as in Example 2 (2). As a result, as shown in FIG. 7, it was shown that this enzyme is very stable and highly heat resistant because it retains 50% or more of activity even after 90 minutes of heat treatment at 80 ° C.
[0030]
(4) pH dependence
The pH of 50 mM Tris buffer in 300 μl of enzyme reaction solution consisting of 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 mM TTP, 1 U inorganic pyrophosphatase is adjusted to (pH 2), (pH 4), (pH 6 ), (PH 6.5), (pH 7), (pH 7.5), (pH 8), 0.0135 mg of the purified enzyme obtained in Example 1 was added to the enzyme reaction solution changed to (pH 10). . The enzyme reaction solution was kept at 80 ° C. for 5 minutes to react, and 30 μl was collected and added to 300 μl of 500 mM KH ₂ PO ₄ solution to stop the reaction. The progress of the reaction was measured by HPLC as in Example 2 (2).
As shown in FIG. 8, the activity of this enzyme showed the highest activity at pH 7.5.
[0031]
(5) Diversity of nucleoside triphosphate substrates
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 24 mM Glucose-1-phosphate, 1 U inorganic pyrophosphatase and 1 mM dTTP or 1 mM dATP, 1 mM dGTP, 1 mM dCTP, 1 mM UTP, 1 0.0135 mg of the purified enzyme obtained in Example 1 was added to 300 μl of an enzyme reaction solution composed of mM ATP, 1 mM GTP, and 1 mM CTP. The enzyme reaction solution was kept at 80 ° C. for 5 minutes to react, and 30 μl was collected and added to 300 μl of 500 mM KH ₂ PO ₄ solution to stop the reaction. The progress of the reaction was measured by HPLC as in Example 2 (2). The results are shown in Table 1. As is apparent from Table 1, it was shown that this enzyme can use dATP, dGTP, dCTP, and UTP as substrates in addition to dTTP.
[0032]
[Table 1]
Use of various nucleoside triphosphates as substrates

[0033]
(6) Diversity of sugar monophosphate substrates
50 mM Tris buffer (pH 7.5), 12 mM MgCl ₂ , 1 mM TTP, 1 U inorganic pyrophosphatase and 24 mM α-D-Glucose-1-phosphate or 24 mM D-fructose-1-phosphate, 24 mM α 0.0135 mg of the purified enzyme obtained in Example 1 was added to 300 μl of an enzyme reaction solution composed of -D-mannose-1-phosphate and 24 mM α-D-Galactose-1-phosphate. The enzyme reaction solution was kept at 80 ° C. for 5 minutes to react, and 30 μl was collected and added to 300 μl of 500 mM KH ₂ PO ₄ solution to stop the reaction. The progress of the reaction was measured by HPLC as in Example 2 (2). As shown in Table 2, it was shown that this enzyme can use D-fructose-1-phosphate and α-D-mannose-1-phosphate as substrates in addition to α-D-Glucose-1-phosphate.
[0034]
[Table 2]
Availability of each sugar monophosphate as a substrate

[0035]
【The invention's effect】
According to the present invention, a novel sugar nucleotide synthase capable of synthesizing various types of sugar nucleotides in a test tube and stable to heat or the like can be provided. As a result, novel sugar nucleotide synthesis has become possible.
On the other hand, sugar nucleotides function as sugar donors in the synthesis of sugar chains of glycoproteins, glycolipids, and polysaccharides. These sugar chain synthesis is closely related to cancer metastasis, organ development or cellular immunity. In recent years, the present invention has attracted attention, and the contribution of the present invention to the development of these studies is extremely large.
[0036]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a graph obtained by measuring separation patterns of TTP and a mixture of TTP and TDP-Glucose by HPLC.
FIG. 2 is a diagram showing a calibration curve of TDP-Glucose using HPLC.
FIG. 3 is a photograph showing an SDS-PAGE pattern of purified STRmlA1 protein.
FIG. 4 shows the amino acid sequence of each protein encoded by the .RmlA protein of M. tuberculosis and the enzyme gene of the present invention (ST0452, ST1971, ST2352), and the sequence motif conserved between these proteins. FIG.
(The number indicates the number of residues from the protein amino group terminal side.)
FIG. 5 is a graph showing sugar nucleotide synthesis activity of STRmlA1 protein at 37 ° C.
FIG. 6 is a graph showing the sugar nucleotide synthesis activity of STRmlA1 protein at 80 ° C.
FIG. 7 is a graph showing the amount of residual activity of STRmlA1 protein after 80 ° C. treatment.
FIG. 8 is a diagram showing changes in activity of STRmlA1 protein due to pH differences.

Claims (1)

As the following, the sugar monophosphate consisting of (a) and the nucleoside triphosphate consisting of (b) are used as substrates (except when glucose-1-phosphate and TTP (thymidine triphosphate ) are used as substrates),
(A) Glucose-1-phosphate, mannose-1-phosphate, or fructose-1-phosphate (b) TTP (thymidine triphosphate), dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate) Fate), dCTP (deoxycytidine triphosphate), or UTP (uridine triphosphate).
A protein having the amino acid sequence described in SEQ ID NO: 4 or an amino acid sequence in which one or more amino acid residues are deleted, substituted, inserted or added in the amino acid sequence described in SEQ ID NO: 4; A method for producing a sugar nucleotide, which comprises allowing a protein having the protein to act . "

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