JPH05317058A - Dna fragment having heat-resistant dna polymerase gene information and its use - Google Patents
Dna fragment having heat-resistant dna polymerase gene information and its useInfo
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- JPH05317058A JPH05317058A JP12464392A JP12464392A JPH05317058A JP H05317058 A JPH05317058 A JP H05317058A JP 12464392 A JP12464392 A JP 12464392A JP 12464392 A JP12464392 A JP 12464392A JP H05317058 A JPH05317058 A JP H05317058A
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- ala
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- gag
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Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、サーマス サーモフィ
ラス(Thermus thermophilus)由来の耐熱性DNAポリ
メラーゼをコードするDNA、及び該DNAを含有する
組換えベクター、該組換えベクターを用いて得られる形
質転換体及び該形質転換体により該DNAの遺伝情報を
発現せしめて得られる耐熱性DNAポリメラーゼの製造
方法に関する。TECHNICAL FIELD The present invention relates to a DNA encoding a thermostable DNA polymerase derived from Thermus thermophilus, a recombinant vector containing the DNA, and a transformant obtained by using the recombinant vector. And a method for producing a thermostable DNA polymerase obtained by expressing the genetic information of the DNA by the transformant.
【0002】[0002]
【従来の技術】近年、ポリメラーゼ チェーン リアク
ション法(PCR法,サイキ等Science230
巻、1350−1354頁(1985年))の普及およ
び、より解析の困難なDNAの塩基配列の解析のために
耐熱性DNAポリメラーゼに対する需要が増大してい
る。しかるに、従来知られているサーマス アクアティ
カス(Thermus aquaticus )由来の耐熱性DNAポリメ
ラーゼ(Taq ポリメラーゼ)は耐熱性が充分ではなく、
反応中に活性の低下が起こる問題があった。そこで我々
はTaq ポリメラーゼよりも熱安定性に優れた耐熱性DN
Aポリメラーゼを見いだし特願昭63−213330を
出願した。2. Description of the Related Art In recent years, the polymerase chain reaction method (PCR method, Saiki etc. Science230
Volume 1350-1354 (1985)) and the demand for thermostable DNA polymerases is increasing due to the analysis of DNA base sequences that are more difficult to analyze. However, the conventionally known thermostable DNA polymerase (Taq polymerase) derived from Thermus aquaticus does not have sufficient thermostability,
There was a problem that the activity decreased during the reaction. Therefore, we used a thermostable DN, which is more thermostable than Taq polymerase.
He found A polymerase and applied for Japanese Patent Application No. 63-213330.
【0003】[0003]
【発明が解決しようとする課題】しかしながら、特願昭
63ー213330で見いだした耐熱性DNAポリメラ
ーゼ生産菌、例えばサーマス サーモフィラス HB8
(ATCC27634)は耐熱性DNAポリメラーゼの
生産性が低く、又制限酵素TthHB8も合わせて生産
するため、精製が煩雑になり、研究用試薬として広く用
いるには必ずしも満足の行くものではなかった。However, a thermostable DNA polymerase-producing bacterium found in Japanese Patent Application No. 63-213330, for example, Thermus thermophilus HB8.
(ATCC 27634) has low productivity of thermostable DNA polymerase, and since it also produces the restriction enzyme TthHB8, purification becomes complicated, and it is not always satisfactory for wide use as a research reagent.
【0004】[0004]
【課題を解決するための手段】本発明者らは該耐熱性D
NAポリメラーゼの生産性の向上を計るべく鋭意検討し
た結果、耐熱性DNAポリメラーゼ遺伝子の採取、なら
びに一次構造解析に成功すると共に、遺伝子工学的手法
を応用することによって、以下に述べるごとく、高生産
性である製法を確立した。The present inventors have proposed the heat resistance D
As a result of diligent studies aimed at improving the productivity of NA polymerase, the thermostable DNA polymerase gene was successfully collected and the primary structure was analyzed, and by applying genetic engineering techniques, high productivity was obtained as described below. Established the manufacturing method.
【0005】すなわち、本発明の要旨はサーマス サー
モフィラス(Thermus thermophirus)由来の耐熱性DN
Aポリメラーゼ又は該耐熱性DNAポリメラーゼと機能
的同等物をコードしているDNA、該DNAを含有する
組換えベクター、宿主細胞を該組換えベクターで形質転
換した形質転換体、及び該形質転換体を培養して耐熱性
DNAポリメラーゼを採取することを特徴とする耐熱性
DNAポリメラーゼの製造法に存する。That is, the gist of the present invention is the thermostable DN derived from Thermus thermophirus.
DNA encoding A polymerase or a functional equivalent to the thermostable DNA polymerase, a recombinant vector containing the DNA, a transformant obtained by transforming a host cell with the recombinant vector, and the transformant A method for producing a thermostable DNA polymerase is characterized in that the thermostable DNA polymerase is collected by culturing.
【0006】本発明の上記DNAとしては、例えば配列
表の配列番号1に記載された塩基配列を有するDNAを
挙げることができる。なお、本発明のDNAは遺伝子組
み換え技術により、基本となるDNAの特定部位に、該
DNAがコードするものの基本的な特性を変化させるこ
となく、あるいはその特性を改善するように人為的に変
異を起こさせたものも含むものである。Examples of the above-mentioned DNA of the present invention include DNA having the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. The DNA of the present invention may be artificially mutated at a specific site of the basic DNA by gene recombination technology without changing the basic characteristics of the one encoded by the DNA or to improve the characteristics. It also includes those that are awakened.
【0007】本発明のDNAは、例えば耐熱性DNAポ
リメラーゼを生産する耐熱性DNAポリメラーゼ遺伝子
の供与体である微生物より、該微生物のDNAを分離・
精製した後、超音波,制限酵素などを用いてDNAを切
断したものとリニヤーな発現ベクターとを両DNAの平
滑または接着末端部においてDNAリガーゼなどにより
結合閉環させ、こうして得られた組み換えDNAベクタ
ーを複製可能な宿主微生物に移入した後、ベクターのマ
ーカーと耐熱性DNAポリメラーゼの活性とを指標とし
て、スクリーニングして取得した該組み換えDNAベク
ターを保持する微生物を培養し、該培養菌体から該組み
換えDNAベクターを分離・精製し、次いで該組み換え
DNAベクターから耐熱性DNAポリメラーゼ遺伝子で
あるDNAを採取すればよい。The DNA of the present invention is obtained by separating the DNA of the microorganism from a microorganism that is a donor of the thermostable DNA polymerase gene producing thermostable DNA polymerase.
After purification, the digested DNA using ultrasonic waves, restriction enzymes, etc. and the linear expression vector are ligated and closed with a DNA ligase at the blunt or cohesive ends of both DNAs, and the recombinant DNA vector thus obtained is After transfer into a replicable host microorganism, the recombinant DNA vector carrying the screened microorganism is cultured using the marker of the vector and the activity of thermostable DNA polymerase as indicators, and the recombinant DNA is recovered from the cultured cells. The vector may be separated and purified, and then the DNA which is a thermostable DNA polymerase gene may be collected from the recombinant DNA vector.
【0008】以下に上記採取方法をより詳細に説明す
る。遺伝子の供与体である微生物に由来するDNAは次
の如くにして採取される。すなわち、供与微生物である
上述した細菌のいずれかを例えば液体培地で約1〜3日
間通気撹拌培養し、得られる培養物を遠心分離して集菌
し、次いでこれを溶菌させることによって耐熱性DNA
ポリメラーゼ遺伝子の含有溶菌物を調製することができ
る。溶菌方法としては例えばリゾチームなどの細胞壁溶
解酵素による処理が施され、必要によりプロテアーゼな
どの他の酵素やラウリル硫酸ナトリウムなどの界面活性
剤が併用され、さらに細胞壁の物理的破壊法である凍結
融解やフレンチプレス処理を上述の溶菌法との組み合せ
で行ってもよい。The above sampling method will be described in more detail below. DNA derived from a microorganism that is a gene donor is collected as follows. That is, any of the above-mentioned bacteria that are donor microorganisms is cultivated by aeration and stirring in a liquid medium for about 1 to 3 days, the resulting culture is centrifuged to collect the cells, and then the cells are lysed to obtain a thermostable DNA.
A lysate containing the polymerase gene can be prepared. As a lysing method, for example, treatment with a cell wall lysing enzyme such as lysozyme is performed, if necessary, other enzymes such as protease and a surfactant such as sodium lauryl sulfate are used in combination, and further, lysing and thawing, which is a method of physically destroying the cell wall, The French press treatment may be performed in combination with the above-mentioned lysis method.
【0009】この様にして得られた溶菌物からDNAを
分離・精製するには常法に従って例えばフェノール抽出
による除蛋白処理,プロテアーゼ処理,リボヌクレアー
ゼ処理,アルコール沈澱遠心分離などの方法を適宜組み
合わせることにより行うことができる。In order to separate and purify DNA from the lysate thus obtained, a method such as deproteinization treatment by phenol extraction, protease treatment, ribonuclease treatment, and alcohol precipitation centrifugation is appropriately combined according to a conventional method. It can be carried out.
【0010】分離・精製された微生物DNAを切断する
方法は、例えば超音波処理,制限酵素処理などにより行
うことができるが、得られるDNA断片とベクターとの
結合を容易ならしめるため制限酵素とりわけ特定ヌクレ
オチド配列に作用する例えばEcoRI,HindII
I,BamHIなどのII型制限酵素が適している。The method of cleaving the separated and purified microbial DNA can be carried out, for example, by ultrasonic treatment or restriction enzyme treatment. However, in order to facilitate the ligation of the obtained DNA fragment and the vector, a restriction enzyme is particularly specified. Acts on nucleotide sequences, eg EcoRI, HindII
Type II restriction enzymes such as I and BamHI are suitable.
【0011】ベクターとしては、宿主微生物で自律的に
増殖しうるファージまたはプラスミドから遺伝子組み換
え用として構築されたものが適している。ファージとし
ては、例えばエシェリシア コリー(Escheric
hiacoli)を宿主微生物とする場合には、λgt
・10,λgt・11などが使用できる。Suitable vectors are those constructed for gene recombination from phages or plasmids capable of autonomous growth in host microorganisms. Examples of phages include Escherichia coli (Escheric)
hictoli) is used as a host microorganism, λgt
· 10, λgt · 11, etc. can be used.
【0012】また、プラスミドとしては、例えばエシェ
リシア コリーを宿主微生物とする場合にはpBR32
2,pBR325,pACYCI84,pUC12,p
UC13,pUC18,pUC19、pKK223−3
などが使用できる。このようなベクターを、先に述べた
耐熱性DNAポリメラーゼ遺伝子供与体である微生物D
NAの切断に使用した制限酵素と同じ制限酵素で切断し
て、ベクター断片を得ることが望ましい。As the plasmid, for example, when Escherichia coli is used as a host microorganism, pBR32
2, pBR325, pACYCI84, pUC12, p
UC13, pUC18, pUC19, pKK223-3
Etc. can be used. Such a vector is prepared by using the above-mentioned microorganism D which is a thermostable DNA polymerase gene donor.
It is desirable to obtain a vector fragment by cutting with the same restriction enzyme as that used for cutting NA.
【0013】微生物DNA断片とベクター断片とを結合
させる方法は、公知のDNAリガーゼを用いる方法であ
ればよく、例えば微生物DNA断片の接着末端とベクタ
ー断片の接着末端とのアニーリングの後、適当なDNA
リガーゼの使用により微生物DNA断片とベクター断片
との組み換えDNAを作成する。必要ならば、アニーリ
ングの後、宿主微生物に移入して、生体内のDNAリガ
ーゼを利用し、組み換えDNAを作成することもでき
る。The method for ligating the microbial DNA fragment and the vector fragment may be a method using a known DNA ligase. For example, after annealing the cohesive ends of the microbial DNA fragment and the vector fragment, a suitable DNA is prepared.
Recombinant DNA of microbial DNA fragment and vector fragment is prepared by using ligase. If necessary, it is also possible to transfer to a host microorganism after annealing and utilize in vivo DNA ligase to prepare a recombinant DNA.
【0014】宿主微生物としては、組み換えDNAが安
定かつ自律的に増殖可能で、且つ外来性DNAの形質が
発現のできるものであればよく、例えば宿主微生物がエ
シェリシア コリーDH5,エシェリシア コリーHB
101,エシェリシア コリーW3110,エシェリシ
ア コリーC600,エシェリシア コリーJM109
などが利用できる。The host microorganism may be any as long as the recombinant DNA can be stably and autonomously propagated and the trait of the foreign DNA can be expressed. For example, the host microorganism is Escherichia coli DH5, Escherichia coli HB.
101, Escherichia Collie W3110, Escherichia Collie C600, Escherichia Collie JM109
Etc. can be used.
【0015】宿主微生物に組み換えDNAを移入する方
法としては、例えば宿主微生物がエシェリシア属に属す
る微生物の場合には、市販のコンピーテントセルを用い
て組み換えDNAの移入を行い、さらにマイクロインジ
ェクション法を用いても良い。こうして得られた形質転
換体である微生物は、栄養培地で培養されることにより
多量の耐熱性DNAポリメラーゼを安定して産生し得る
ことを見出した。As a method for transferring the recombinant DNA into the host microorganism, for example, when the host microorganism is a microorganism belonging to the genus Escherichia, the recombinant DNA is transferred using a commercially available competent cell, and then the microinjection method is used. May be. It was found that the thus obtained transformant microorganism can stably produce a large amount of thermostable DNA polymerase when cultured in a nutrient medium.
【0016】宿主微生物への目的組み換えDNA移入の
有無についての選択は、目的組み換えDNAを保持する
ベクターの薬剤耐性マーカーと耐熱性DNAポリメラー
ゼとを同時に発現し得る微生物を検索する方法、選択培
地に成育したコロニーについて、アミノ酸配列を基に合
成したDNAプローブとハイブリザイゼーションを行い
検索する方法等があげられる。The selection as to whether or not the target recombinant DNA has been transferred into the host microorganism is carried out by a method of searching for a microorganism capable of simultaneously expressing a drug resistance marker of a vector carrying the target recombinant DNA and a thermostable DNA polymerase, and growing it in a selective medium. The colonies thus formed may be hybridized with a DNA probe synthesized on the basis of the amino acid sequence to perform a search and the like.
【0017】上述の方法により得られた耐熱性DNAポ
リメラーゼ遺伝子の塩基配列はサイエンス(Scien
ce),214,1205〜1210(1981)に記
載されているジデオキシ法で解読し、また耐熱性DNA
ポリメラーゼ遺伝子の全アミノ酸配列は塩基配列より推
定した。この様にして一度選択された耐熱性DNAポリ
メラーゼ遺伝子を保有する組み換えDNAは、形質転換
微生物から取り出され、他の宿主微生物に移入すること
も容易に実施できる。また、耐熱性DNAポリメラーゼ
遺伝子を保持する組み換えDNAから制限酵素などによ
り切断して、耐熱性DNAポリメラーゼ遺伝子であるD
NAを切り出し、これを同様な方法により切断して得ら
れるベクター断片とを結合させて、宿主微生物に移入す
ることも容易に実施できる。The base sequence of the thermostable DNA polymerase gene obtained by the above method is determined by Science (Scien).
ce), 214, 1205-1210 (1981), deciphering by the dideoxy method, and thermostable DNA
The entire amino acid sequence of the polymerase gene was deduced from the nucleotide sequence. The recombinant DNA carrying the thermostable DNA polymerase gene once selected in this manner can be easily taken out from the transformed microorganism and transferred to another host microorganism. In addition, a thermostable DNA polymerase gene D, which is a thermostable DNA polymerase gene, is cleaved from a recombinant DNA carrying the thermostable DNA polymerase gene by a restriction enzyme.
It is also easy to cut out NA, ligate it with a vector fragment obtained by cutting it by the same method, and transfer it to a host microorganism.
【0018】形質転換体である宿主微生物の培養形態は
宿主の栄養生理的性質を考慮して培養条件を選択すれば
良く、通常多くの場合は液体培養で行うが、工業的には
通気撹拌培養を行うのが有利である。培地の栄養源とし
ては、微生物の培養に通常用いられるものが広く使用さ
れ得る。炭素源としては資化可能な炭素化合物であれば
よく、例えばグルコ−ス,シュクロース,ラクトース,
マルトース,フラクトース,糖蜜,ピルビン酸などが使
用される。窒素源としては利用可能な窒素化合物であれ
ばよく、例えばペプトン,肉エキス,酵母エキス,カゼ
イン加水分解物,大豆粕アルカリ抽出物などが使用され
る。その他、リン酸塩,炭酸塩,硫酸塩,マグネシウ
ム,カルシウム,カリウム,鉄,マンガン,亜鉛などの
塩類,特定のアミノ酸,特定のビタミンなどが必要に応
じて使用される。The culturing form of the host microorganism as a transformant may be selected by considering the culturing conditions in consideration of the nutritional physiological properties of the host. Usually, liquid culturing is usually carried out. Is advantageous. As the nutrient source of the medium, those usually used for culturing microorganisms can be widely used. The carbon source may be any assimilable carbon compound, for example, glucose, sucrose, lactose,
Maltose, fructose, molasses, pyruvic acid, etc. are used. Any available nitrogen compound may be used as the nitrogen source, and for example, peptone, meat extract, yeast extract, casein hydrolyzate, soybean meal alkali extract and the like are used. In addition, salts such as phosphates, carbonates, sulfates, magnesium, calcium, potassium, iron, manganese, zinc, specific amino acids, specific vitamins, etc. are used as necessary.
【0019】培養温度は菌が発育し、耐熱性DNAポリ
メラーゼを生産する範囲で適宜変更し得るが、エシェリ
シア コリーの場合、好ましくは20〜42℃程度であ
る。培養時間は条件によって多少異なるが、耐熱性DN
Aポリメラーゼが最高収量に達する時期を見計らって適
当な時期に培養を終了すれば良く、通常は12〜48時
間程度である。培地pHは菌が発育し、耐熱性DNAポ
リメラーゼを生産する範囲で適宜変更し得るが、特に好
ましくはpH6.0〜8.0程度である。The culturing temperature may be appropriately changed within the range where the bacterium grows and produces a thermostable DNA polymerase, but in the case of Escherichia coli, it is preferably about 20 to 42 ° C. Culture time varies slightly depending on the conditions, but heat-resistant DN
The culture may be completed at an appropriate time, considering the time when the maximum yield of A polymerase is reached, and usually about 12 to 48 hours. The pH of the medium can be appropriately changed within the range in which the bacterium grows and the thermostable DNA polymerase is produced, but the pH is particularly preferably about 6.0 to 8.0.
【0020】培養物中の耐熱性DNAポリメラーゼは、
培養物を濾過または遠心分離などの手段により、菌体を
採取し次いでこの菌体を機械的方法またはリゾチームな
どの酵素的方法で破壊し、また必要に応じてEDTA等
のキレート剤および/または界面活性剤を添加して耐熱
性DNAポリメラーゼを可溶化し、水溶液として分離採
取する。The thermostable DNA polymerase in the culture is
The culture is harvested by a means such as filtration or centrifugation, and then the cells are disrupted by a mechanical method or an enzymatic method such as lysozyme, and if necessary, a chelating agent such as EDTA and / or an interface. The thermostable DNA polymerase is solubilized by adding an activator and separated and collected as an aqueous solution.
【0021】この様にして得られた耐熱性DNAポリメ
ラーゼ含有溶液を、例えば減圧濃縮,膜濃縮,更に硫酸
アンモニウム,硫酸ナトリウムなどの塩析処理、あるい
は親水性有機溶媒、例えばメタノール,エタノール,ア
セトンなどによる分別沈澱法により沈澱せしめればよ
い。また、加熱処理や等電点処理も有効な精製手段であ
る。吸着剤あるいはゲル濾過剤などによるゲル濾過,吸
着クロマトグラフィー,イオン交換クロマトグラフィ
ー,アフィニティークロマトグラフィーにより、精製さ
れた耐熱性DNAポリメラーゼを得る。The heat-resistant DNA polymerase-containing solution thus obtained is subjected to, for example, vacuum concentration, membrane concentration, salting out with ammonium sulfate, sodium sulfate or the like, or with a hydrophilic organic solvent such as methanol, ethanol or acetone. It may be precipitated by the fractional precipitation method. Further, heat treatment and isoelectric point treatment are also effective refining means. A purified thermostable DNA polymerase is obtained by gel filtration using an adsorbent or a gel filtration agent, adsorption chromatography, ion exchange chromatography, and affinity chromatography.
【0022】以下、実施例で本発明を詳細に説明する。The present invention will be described in detail below with reference to examples.
実施例1〔染色体DNAの分離〕 サーマス サーモフィラスHB8の染色体DNAを次の
方法で分離した。同菌株を150mlの1.5%ポリペ
プトン、1.5%酵母エキス、0.2%NaCl(pH7.
5)よりなる液体培地で70℃一晩振盪培養後、遠心
(8000rpm,10分)により集菌した。10mM
トリス塩酸(pH8.0)、1mM EDTAを含んだ
溶液24mlに懸濁させ、3mlのリゾチーム溶液(2
0mg/ml)を加えて、その後10%SDS溶液を6
ml加えた。30mlのフェノールを加えてゆるやかに
撹拌混合し、10,000rpm,15分間の遠心で水
層と溶媒層に分け、水層を合取した。この操作を再びお
こない、この水層に2倍量のエタノールを静かに重層
し、ガラス棒でゆっくり撹拌しながらDNAをガラス棒
にまきつかせて分離した。これを10mMトリス塩酸
(pH8.0)、1mM EDTAを含んだ溶液(以下
TEと記載)で溶解した。これを等量のクロロホルム・
フェノール溶液で処理後、遠心により水層を分取し、2
倍量のエタノールを加えて、上記の方法でもう一度DN
Aを分離し2mlのTEで溶解した。エシェリシア コ
リーJM109のコンピテントセルは市販(東洋紡社
製)のものを使用し、ライブラリー作成の宿主として用
いた。Example 1 [Separation of chromosomal DNA] The chromosomal DNA of Thermus thermophilus HB8 was separated by the following method. 150 ml of 1.5% polypeptone, 1.5% yeast extract, 0.2% NaCl (pH 7.
After culturing with shaking in a liquid medium consisting of 5) at 70 ° C. overnight, the cells were collected by centrifugation (8000 rpm, 10 minutes). 10 mM
Tris-hydrochloric acid (pH 8.0), suspended in 24 ml of a solution containing 1 mM EDTA, and suspended in 3 ml of a lysozyme solution (2
0 mg / ml) and then 6% 10% SDS solution.
ml was added. 30 ml of phenol was added, and the mixture was gently mixed with stirring, centrifuged at 10,000 rpm for 15 minutes to separate an aqueous layer and a solvent layer, and the aqueous layers were combined. This operation was performed again, 2 volumes of ethanol was gently layered on this aqueous layer, and the DNA was sprinkled on the glass rod while gently stirring with the glass rod to separate the DNA. This was dissolved in a solution containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA (hereinafter referred to as TE). Add an equal amount of chloroform
After treatment with phenol solution, separate the aqueous layer by centrifugation and
Add twice the amount of ethanol and repeat the above procedure using DN.
A was separated and dissolved with 2 ml TE. A commercially available competent cell of Escherichia coli JM109 (manufactured by Toyobo Co., Ltd.) was used and used as a host for preparing a library.
【0023】実施例2〔耐熱性DNAポリメラーゼをコ
ードする遺伝子を含有するDNA断片及び該DNA断片
を有する組み換えベクターの調製〕 実施例1で得たDNA1μgを制限酵素HindIII
(東洋紡製)5ユニットで37℃,15分反応させ、限
定分解を行った後、制限酵素HindIIIにて完全に
切断したpUC19 0.5μgとT4 −DNAリガー
ゼ(東洋紡製)1ユニットで16℃、12時間反応させ
DNAを連結した。連結したDNAは、エシェリシア
コリーJM109のコンピテントセルを用いて形質転換
した。使用したDNA1μg当り約1×106 個の形質
転換体のコロニーが得られた。得られたコロニーは50
μg/mlアンピシリン入りL培地(1%ポリペプト
ン,0.5%酵母エキス,0.5%塩化ナトリウム、
0.25mMIPTG)で37℃で18時間培養し、菌
体を破砕し、75℃、30分処理した液を用いて以下の
方法により耐熱性DNAポリメラーゼ活性を測定した。Example 2 [Preparation of a DNA fragment containing a gene encoding a thermostable DNA polymerase and a recombinant vector having the DNA fragment] 1 μg of the DNA obtained in Example 1 was used as a restriction enzyme HindIII.
(Toyobo) 5 units were allowed to react at 37 ° C. for 15 minutes, limited digestion was performed, and 0.5 μg of pUC19 completely digested with the restriction enzyme HindIII and 1 unit of T 4 -DNA ligase (Toyobo) were used at 16 ° C. The reaction was carried out for 12 hours to ligate the DNA. The ligated DNA is Escherichia
Transformation was performed using competent cells of Collie JM109. About 1 × 10 6 transformant colonies were obtained per 1 μg of the DNA used. 50 colonies obtained
L medium containing μg / ml ampicillin (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride,
The cells were cultivated in 0.25 mM IPTG) at 37 ° C. for 18 hours, cells were crushed, and the heat-resistant DNA polymerase activity was measured by the following method using the solution treated at 75 ° C. for 30 minutes.
【0024】(試薬) A.670mM Tris-HCl(pH8.8) 166mM 硫酸アンモニウム 67mM 塩化マグネシウム 10mM 2−メルカプトエタノール B. 1μg/μl 活性化仔牛胸線DNA C. 2mM dNTP(250cpm/ pmol
「3H]dTTP) D. 20% トリクロロ酢酸(2mM ピロリン
酸) E. 1μg/μl キャリアーDNA(Reagent) A. 670 mM Tris-HCl (pH 8.8) 166 mM ammonium sulfate 67 mM magnesium chloride 10 mM 2-mercaptoethanol B.I. 1 μg / μl Activated Calf Thorax DNA C. 2 mM dNTP (250 cpm / pmol
“3H] dTTP) D. 20% trichloroacetic acid (2 mM pyrophosphate) E. 1 μg / μl carrier DNA
【0025】(方法)A液、B液、C液各5μl 、及び
滅菌水30μl をエッペンドルフチューブに加えて撹拌
混合後、菌体破砕液、あるいは上記熱処理液5μl を加
え、75℃で30分間反応する。その後氷冷しE液50
μl 、D液100mlを加え、更に10分間氷冷する。
この液をガラスフィルター(ワットマンGF/Cフィル
ター)で濾過しD液及び冷エタノールで充分洗浄し、フ
ィルターの放射活性を液体シンチレーションカウンター
(パッカード社製)で計測し、鋳型DNAへのヌクレオ
チドの取り込みを測定した。 酵素活性の1単位は、こ
の条件下で30分間当り10nmolのヌクレオチドを
酸不溶性画分に取り込む酵素量とした。(Method) Solution A, solution B and solution C (5 μl each) and sterilized water (30 μl) were added to an Eppendorf tube and mixed by stirring, and then the cell disruption solution or the heat treatment solution (5 μl) was added and reacted at 75 ° C. for 30 minutes. To do. Then ice-cool and E-solution 50
Add μl and 100 ml of solution D, and cool with ice for 10 minutes.
This solution was filtered with a glass filter (Whatman GF / C filter), thoroughly washed with solution D and cold ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard) to incorporate nucleotides into the template DNA. It was measured. One unit of enzyme activity was the amount of enzyme that incorporated 10 nmol of nucleotide into the acid-insoluble fraction per 30 minutes under these conditions.
【0026】上記方法により約3,000個のコロニー
の耐熱性DNAポリメラーゼ活性を測定したところ、2
個の耐熱性DNAポリメラーゼ活性を有するコロニーを
得た。これらの保有するプラスミドには内部にHind
IIIサイトを1ケ 所含む約6kbpの挿入DNA断片
が存在しており、このプラスミドをpUPL0とした。
次いでpUPL0よりHindIIIにて約2.8kb
p、及び3.2kbpの挿入DNA断片を切り出し、そ
れぞれpUC19へ導入してpUPL01及びpUPL
02を構築した。それぞれをE.coliJM109 に形質転換
し、この形質転換体を培養後上記方法により耐熱性DN
Aポリメラーゼ活性を測定したところ活性は認められな
かった。次に pUPL02をPstI、HindII
I処理して得られる1.4kbpの断片をpUC19に
導入し、pUPL03を構築した。更にpUPL01を
HindIII処理して得られる3.2kbpの断片を
pUPL03のHindIIIサイトに導入しpUPL
1を構築した。pUPL1の構築方法を図1に示す。こ
のpUPL1でエシェリシア コリーJM109を形質
転換した菌株の耐熱性DNAポリメラーゼの生産性はサ
ーマス サーモフィラスHB8の約2倍であった。When the thermostable DNA polymerase activity of about 3,000 colonies was measured by the above method, 2
Colonies having thermostable DNA polymerase activity were obtained. These plasmids have Hind inside.
An insertion DNA fragment of about 6 kbp containing one III site was present, and this plasmid was designated as pUPLO.
Then from pUPL0, about 2.8kb in HindIII
The inserted DNA fragments of p and 3.2 kbp were excised and introduced into pUC19 to obtain pUPL01 and pUPL, respectively.
02 was built. Each of them was transformed into E. coli JM109, the transformants were cultured, and then heat-resistant DN was obtained by the above-mentioned method.
When the A polymerase activity was measured, no activity was observed. Next, pUPL02 is replaced with PstI and HindII
The 1.4 kbp fragment obtained by treating with I was introduced into pUC19 to construct pUPL03. Furthermore, a 3.2 kbp fragment obtained by treating pUPL01 with HindIII was introduced into the HindIII site of pUPL03 to obtain pUPL.
Built 1. The construction method of pUPL1 is shown in FIG. The productivity of the thermostable DNA polymerase of the strain transformed with Escherichia coli JM109 with pUPL1 was about twice that of Thermus thermophilus HB8.
【0027】[0027]
【表1】 [Table 1]
【0028】実施例3〔塩基配列の決定〕 pUPL01の約2.8kbpのHindIII断片、
及びpUPL03の約1.4kbpのHindIII-
PstI断片について、TAKARA kilo se
quence deletion kitを用いてde
letionmutantの作製を行った。delet
ion mutantは常法に従い一本鎖DNAとし、
得られた一本鎖DNAについて、Tthシークエンシン
グキット(Tth Sequen−cing kit、
東洋紡製)を用いて、塩基配列の決定を行った。決定し
た塩基配列及びアミノ酸配列を配列1に示した。Example 3 [Determination of nucleotide sequence] A HindIII fragment of pUPL01 of about 2.8 kbp,
And about 1.4 kbp HindIII-of pUPL03
For the PstI fragment, TAKARA
de using the sequence deletion kit
A production of a leakage mutant was performed. delete
Ion mutant is a single-stranded DNA according to a conventional method,
About the obtained single-stranded DNA, the Tth Sequencing kit (Tth Sequencing-Kit,
The base sequence was determined by using Toyobo. The determined nucleotide sequence and amino acid sequence are shown in Sequence 1.
【0029】[0029]
【発明の効果】本発明によって、耐熱性DNAポリメラ
ーゼ遺伝子の塩基配列および耐熱性DNAポリメラーゼ
のアミノ酸配列が明らかになり、また遺伝子工学的手法
による効率的な耐熱性DNAポリメラーゼの製造法を提
供した。また本発明の耐熱性DNAポリメラーゼ遺伝子
と種々の遺伝子工学的手法とを用いることにより、より
効率的な耐熱性DNAポリメラーゼの製造法をもたらし
めるものである。INDUSTRIAL APPLICABILITY The present invention clarified the base sequence of a thermostable DNA polymerase gene and the amino acid sequence of a thermostable DNA polymerase, and provided an efficient method for producing a thermostable DNA polymerase by a genetic engineering technique. Further, by using the thermostable DNA polymerase gene of the present invention and various genetic engineering techniques, a more efficient method for producing the thermostable DNA polymerase can be brought about.
【0030】[0030]
配列番号:1 配列の長さ:3221 配列の型:核酸 鎖の数:二本鎖 トポロジー:直鎖状 配列の種類: genomic DNA 起源 生物名:サーマス サーモフィラス(Thermus thermophi
lus) 株名:HB8(ATCC27634) TCTAGAGGAA GCATGAGCCT CACCCTGGCA GACAAGGTGG TCTACGAGGA GGAGATCCAG 60 AAAAGCCGCT TCATCGCCAA GGCGGCCCCC GTGGCCTCGG AGGAGGAGGC CTTGGCGTTT 120 TTGGCCGAGA ACCGGGAGCC TGAGGCCACC CACAACGGCC ACGCCTACAA GATCGGCCTC 180 CTCTACCGCT TCTCTGACGA CGGGGAGCCC TCGGGCACCG CAGGCAGGCC CATCCTCCAC 240 GCCATAGAGG CCCAGGGCCT GGACCGGGTG GCGGTCCTGG TGGTGCGCTA CTTCGGCGGG 300 GTGAAGCTCG GGGCCGGGGG GCTTGTGCGG GCCTACGGGG GGGTGGCGGC GGAGGCCTTA 360 AGGCGGGCGC CCAAGGTCCC CTTGGTGGAG CGGGTGGGGC TCGCCTTCCT CGTGCCCTTC 420 GCCGAGGTGG GCCGGGTCTA CGCCCTCCTG GAGGCCCGCG CCCCTGAAGG CCGAGGAGAC 480 CTACACCCCG GAGGCGTGCG CTTCGCCCTC CTCCTCCCCA AGCCCGAGCG GGAAGGTTTC 540 CTCAGGGCGC TCCTGGACGC CACCCGGGGA CAGGTGGCCC TGGAGTAGC ATG GAG 595 Met Glu 1 GCG ATG CTT CCG CTC TTT GAA CCC AAA GGC CGG GTC CTC CTG GTG GAC 643 Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val Asp 5 10 15 GGC CAC CAC CTG GCC TAC CGC ACC TTC TTC GCC CTG AAG GGC CTC ACC 691 Gly His His Leu Ala Tyr Arg Thr Phe Phe Ala Leu Lys Gly Leu Thr 20 25 30 ACG AGC CGG GGC GAA CCG GTG CAG GCG GTC TAC GGC TTC GCC AAG AGC 739 Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys Ser 35 40 45 50 CTC CTC AAG GCC CTG AAG GAG GAC GGG TAC AAG GCC GTC TTC GTG GTC 787 Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe Val Val 55 60 65 TTT GAC GCC AAG GCC CCC TCC TTC CGC CAC GAG GCC TAC GAG GCC TAC 835 Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr 70 75 80 AAG GCG GGG AGG GCC CCG ACC CCC GAG GAC TTC CCC CGG CAG CTC GCC 883 Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala 85 90 95 CTC ATC AAG GAG CTG GTG GAC CTC CTG GGG TTT ACC CGC CTC GAG GTC 931 Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu Glu Val 100 105 110 CCC GGC TAC GAG GCG GAC GAC GTT CTC GCC ACC CTG GCC AAG AAG GCG 979 Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Lys Ala 115 120 125 130 GAA AAG GAG GGG TAC GAG GTG CGC ATC CTC ACC GCC GAC CGC GAC CTC 1027 Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Arg Asp Leu 135 140 145 TAC CAA CTC GTC TCC GAC CGC GTC GCC GTC CTC CAC CCC GAG GGC CAC 1075 Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly His 150 155 160 CTC ATC ACC CCG GAG TGG CTT TGG GAG AAG TAC GGC CTC AGG CCG GAG 1123 Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro Glu 165 170 175 CAG TGG GTG GAC TTC CGC GCC CTC GTG GGG GAC CCC TCC GAC AAC CTC 1171 Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp Asn Leu 180 185 190 CCC GGG GTC AAG GGC ATC GGG GAG AAG ACC GCC CTC AAG CTC CTC AAG 1219 Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu Leu Lys 195 200 205 210 GAG TGG GGA AGC CTG GAA AAC CTC CTC AAG AAC CTG GAC CGG GTA AAG 1267 Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val Lys 215 220 225 CCA GAA AAC GTC CGG GAG AAG ATC AAG GCC CAC CTG GAA GAC CTC AGG 1315 Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp Leu Arg 230 235 240 CTC TCC TTG GAG CTC TCC CGG GTG CGC ACC GAC CTC CCC CTG GAG GTG 1363 Leu Ser Leu Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 GAC CTC GCC CAG GGG CGG GAG CCC GAC CGG GAG GGG CTT AGG GCC TTC 1411 Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala Phe 260 265 270 CTG GAG AGG CTG GAG TTC GGC AGC CTC CTC CAC GAG TTC GGC CTC CTG 1459 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 290 GAG GCC CCC GCC CCC CTG GAG GAG GCC CCC TGG CCC CCG CCG GAA GGG 1507 Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 295 300 305 GCC TTC GTG GGC TTC GTC CTC TCC CGC CCC GAG CCC ATG TGG GCG GAG 1555 Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu 310 315 320 CTT AAA GCC CTG GCC GCC TGC AGG GAC GGC CGG GTG CAC CGG GCA GCA 1603 Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg Ala Ala 325 330 335 GAC CCC TTG GCG GGG CTA AAG GAC CTC AAG GAG GTC CGG GGC CTC CTC 1651 Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly Leu Leu 340 345 350 GCC AAG GAC CTC GCC GTC TTG GCC TCG AGG GAG GGG CTA GAC CTC GTG 1699 Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp Leu Val 355 360 365 370 CCC GGG GAC GAC CCC ATG CTC CTC GCC TAC CTC CTG GAC CCC TCC AAC 1747 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 375 380 385 ACC ACC CCC GAG GGG GTG GCG CGG CGC TAC GGG GGG GAG TGG ACG GAG 1795 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 390 395 400 GAC GCC GCC CAC CGG GCC CTC CTC TCG GAG AGG CTC CAT CGG AAC CTC 1843 Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu 405 410 415 CTT AAG CGC CTC GAG GGG GAG GAG AAG CTC CTT TGG CTC TAC CAC GAG 1891 Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu 420 425 430 GTG GAA AAG CCC CTC TCC CGG GTC CTG GCC CAC ATG GAG GCC ACC GGG 1939 Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 450 GTA CGG CGG GAC GTG GCC TAC CTT CAG GCC CTT TCC CTG GAG CTT GCG 1987 Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu Leu Ala 455 460 465 GAG GAG ATC CGC CGC CTC GAG GAG GAG GTC TTC CGC TTG GCG GGC CAC 2035 Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala Gly His 470 475 480 CCC TTC AAC CTC AAC TCC CGG GAC CAG CTG GAA AGG GTG CTC TTT GAC 2083 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 GAG CTT AGG CTT CCC GCC TTG GGG AAG ACG CAA AAG ACA GGC AAG CGC 2131 Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly Lys Arg 500 505 510 TCC ACC AGC GCC GCG GTG CTG GAG GCC CTA CGG GAG GCC CAC CCC ATC 2179 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 530 GTG GAG AAG ATC CTC CAG CAC CGG GAG CTC ACC AAG CTC AAG AAC ACC 2227 Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn Thr 535 540 545 TAC GTG GAC CCC CTC CCA AGC CTC GTC CAC CCG AGG ACG GGC CGC CTC 2275 Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg Leu 550 555 560 CAC ACC CGC TTC AAC CAG ACG GCC ACG GCC ACG GGG AGG CTT AGT AGC 2323 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 TCC GAC CCC AAC CTG CAG AAC ATC CCC GTC CGC ACC CCC TTG GGC CAG 2371 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 AGG ATC CGC CGG GCC TTC GTG GCC GAG GCG GGT TGG GCG TTG GTG GCC 2419 Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val Ala 595 600 605 610 CTG GAC TAT AGC CAG ATA GAG CTC CGC GTC CTC GCC CAC CTC TCC GGG 2467 615 620 625 GAC GAA AAC CTG ATC AGG GTC TTC CAG GAG GGG AAG GAC ATC CAC ACC 2515 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile His Thr 630 635 640 CAG ACC GCA AGC TGG ATG TTC GGC GTC CCC CCG GAG GCC GTG GAC CCC 2563 Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro 645 650 655 CTG ATG CGC CGG GCG GCC AAG ACG GTG AAC TTC GGC GTC CTC TAC GGC 2611 Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly 660 665 670 ATG TCC GCC CAT AGG CTC TCC CAG GAG CTT GCC ATC CCC TAC GAG GAG 2659 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 690 GCG GTG GCC TTT ATA GAG CGC TAC TTC CAA AGC TTC CCC AAG GTG CGG 2707 Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 695 700 705 GCC TGG ATA GAA AAG ACC CTG GAG GAG GGG AGG AAG CGG GGC TAC GTG 2755 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly Tyr Val 710 715 720 GAA ACC CTC TTC GGA AGA AGG CGC TAC GTG CCC GAC CTC AAC GCC CGG 2803 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala Arg 725 730 735 GTG AAG AGC GTC AGG GAG GCC GCG GAG CGC ATG GCC TTC AAC ATG CCC 2851 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG CTC GCC ATG GTG AAG CTC 2899 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 770 Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln Val His 775 780 785 GAC GAG CTC CTC CTG GAG GCC CCC CAA GCG CGG GCC GAG GAG GTG GCG 2995 Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu Val Ala 790 795 800 GCT TTG GCC AAG GAG GCC ATG GAG AAG GCC TAT CCC CTC GCC GTG CCC 3043 Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala Val Pro 805 810 815 CTG GAG GTG GAG GTG GGG ATG GGG GAG GAC TGG CTT TCC GCC AAG GGT 3091 Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala Lys Gly 820 825 830 TAGGGGGGCC CTGCCGTTTA GAGGAAGTTC AAGGGGTTGT CCCTCAGAAA CGCCTCCAGG 3151 GGAACGCCCT CTGGGCCTAC CAGGAGGCCT TTAGCCCCAA AGGTGCGGGT GAAGGCTTCC 3211 AGGCCCTGGG 3221SEQ ID NO: 1 Sequence length: 3221 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Thermus thermophi
lus) strain name: HB8 (ATCC27634) TCTAGAGGAA GCATGAGCCT CACCCTGGCA GACAAGGTGG TCTACGAGGA GGAGATCCAG 60 AAAAGCCGCT TCATCGCCAA GGCGGCCCCC GTGGCCTCGG AGGAGGAGGC CTTGGCGTTT 120 TTGGCCGAGA ACCGGGAGCC TGAGGCCACC CACAACGGCC ACGCCTACAA GATCGGCCTC 180 CTCTACCGCT TCTCTGACGA CGGGGAGCCC TCGGGCACCG CAGGCAGGCC CATCCTCCAC 240 GCCATAGAGG CCCAGGGCCT GGACCGGGTG GCGGTCCTGG TGGTGCGCTA CTTCGGCGGG 300 GTGAAGCTCG GGGCCGGGGG GCTTGTGCGG GCCTACGGGG GGGTGGCGGC GGAGGCCTTA 360 AGGCGGGCGC CCAAGGTCCC CTTGGTGGAG CGGGTGGGGC TCGCCTTCCT CGTGCCCTTC 420 GCCGAGGTGG GCCGGGTCTA CGCCCTCCTG GAGGCCCGCG CCCCTGAAGG CCGAGGAGAC 480 CTACACCCCG GAGGCGTGCG CTTCGCCCTC CTCCTCCCCA AGCCCGAGCG GGAAGGTTTC 540 CTCAGGGCGC TCCTGGACGC CACCCGGGGA CAGGTGGCCC TGGAGTAGC ATG GAG 595 Met Glu 1 GCG ATG CTT CCG CTC TTT GAA CCC AAA GGC CGG GTC CTC CTG GTG GAC 643 Ala Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu Val Asp 5 10 15 GGC CAC CAC CTG GCC TAC CGC ACC TTC TTC GCC CTG AAG GGC CTC ACC 691 Gly His His Leu Ala Tyr Arg Th r Phe Phe Ala Leu Lys Gly Leu Thr 20 25 30 ACG AGC CGG GGC GAA CCG GTG CAG GCG GTC TAC GGC TTC GCC AAG AGC 739 Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala Lys Ser 35 40 45 50 CTC CTC AAG GCC CTG AAG GAG GAC GGG TAC AAG GCC GTC TTC GTG GTC 787 Leu Leu Lys Ala Leu Lys Glu Asp Gly Tyr Lys Ala Val Phe Val Val 55 60 65 TTT GAC GCC AAG GCC CCC TCC TTC CGC CAC GAG GCC TAC GAG GCC TAC 835 Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Glu Ala Tyr 70 75 80 AAG GCG GGG AGG GCC CCG ACC CCC GAG GAC TTC CCC CGG CAG CTC GCC 883 Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu Ala 85 90 95 CTC ATC AAG GAG CTG GTG GAC CTC CTG GGG TTT ACC CGC CTC GAG GTC 931 Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Phe Thr Arg Leu Glu Val 100 105 110 CCC GGC TAC GAG GCG GAC GAC GTT CTC GCC ACC CTG GCC AAG AAG GCG 979 Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Thr Leu Ala Lys Lys Ala 115 120 125 130 GAA AAG GAG GGG TAC GAG GTG CGC ATC CTC ACC GCC GAC CGC GAC CTC 1027 Glu Lys Glu Gly Tyr Glu Val A rg Ile Leu Thr Ala Asp Arg Asp Leu 135 140 145 TAC CAA CTC GTC TCC GAC CGC GTC GCC GTC CTC CAC CCC GAG GGC CAC 1075 Tyr Gln Leu Val Ser Asp Arg Val Ala Val Leu His Pro Glu Gly His 150 155 160 CTC ATC ACC CCG GAG TGG CTT TGG GAG AAG TAC GGC CTC AGG CCG GAG 1123 Leu Ile Thr Pro Glu Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro Glu 165 170 175 CAG TGG GTG GAC TTC CGC GCC CTC GTG GGG GAC CCC TCC GAC AAC CTC 1171 Gln Trp Val Asp Phe Arg Ala Leu Val Gly Asp Pro Ser Asp Asn Leu 180 185 190 CCC GGG GTC AAG GGC ATC GGG GAG AAG ACC GCC CTC AAG CTC CTC AAG 1219 Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Leu Lys Leu Leu Lys 195 200 205 210 GAG TGG GGA AGC CTG GAA AAC CTC CTC AAG AAC CTG GAC CGG GTA AAG 1267 Glu Trp Gly Ser Leu Glu Asn Leu Leu Lys Asn Leu Asp Arg Val Lys 215 220 225 CCA GAA AAC GTC CGG GAG AAG ATC AAG GCC CAC CTG GAA GAC CTC AGG 1315 Pro Glu Asn Val Arg Glu Lys Ile Lys Ala His Leu Glu Asp Leu Arg 230 235 240 CTC TCC TTG GAG CTC TCC CGG GTG CGC ACC GAC CTC CCC CTG GAG GTG 1363 Leu Ser Le u Glu Leu Ser Arg Val Arg Thr Asp Leu Pro Leu Glu Val 245 250 255 GAC CTC GCC CAG GGG CGG GAG CCC GAC CGG GAG GGG CTT AGG GCC TTC 1411 Asp Leu Ala Gln Gly Arg Glu Pro Asp Arg Glu Gly Leu Arg Ala Phe 260 265 270 CTG GAG AGG CTG GAG TTC GGC AGC CTC CTC CAC GAG TTC GGC CTC CTG 1459 Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu 275 280 285 290 GAG GCC CCC GCC CCC CTG GAG GAG GCC CCC TGG CCC CCG CCG GAA GGG 1507 Glu Ala Pro Ala Pro Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly 295 300 305 GCC TTC GTG GGC TTC GTC CTC TCC CGC CCC GAG CCC ATG TGG GCG GAG 1555 Ala Phe Val Gly Phe Val Leu Ser Arg Pro Glu Pro Met Trp Ala Glu 310 315 320 CTT AAA GCC CTG GCC GCC TGC AGG GAC GGC CGG GTG CAC CGG GCA GCA 1603 Leu Lys Ala Leu Ala Ala Cys Arg Asp Gly Arg Val His Arg Ala Ala 325 330 335 GAC CCC TTG GCG GGG CTA AAG GAC CTC AAG GAG GTC CGG GGC CTC CTC 1651 Asp Pro Leu Ala Gly Leu Lys Asp Leu Lys Glu Val Arg Gly Leu Leu 340 345 350 GCC AAG GAC CTC GCC GTC TTG GCC TCG AGG GAG GGG CTA GAC CTC GTG 1699 Ala Lys Asp Leu Ala Val Leu Ala Ser Arg Glu Gly Leu Asp Leu Val 355 360 365 370 CCC GGG GAC GAC CCC ATG CTC CTC GCC TAC CTC CTG GAC CCC TCC AAC 1747 Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn 375 380 385 ACC ACC CCC GAG GGG GTG GCG CGG CGC TAC GGG GGG GAG TGG ACG GAG 1795 Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu 390 395 400 GAC GCC GCC CAC CGG GCC CTC CTC TCG GAG AGG CTC CAT CGG AAC CTC 1843 Asp Ala Ala His Arg Ala Leu Leu Ser Glu Arg Leu His Arg Asn Leu 405 410 415 CTT AAG CGC CTC GAG GGG GAG GAG AAG CTC CTT TGG CTC TAC CAC GAG 1891 Leu Lys Arg Leu Glu Gly Glu Glu Lys Leu Leu Trp Leu Tyr His Glu 420 425 430 GTG GAA AAG CCC CTC TCC CGG GTC CTG GCC CAC ATG GAG GCC ACC GGG 1939 Val Glu Lys Pro Leu Ser Arg Val Leu Ala His Met Glu Ala Thr Gly 435 440 445 450 GTA CGG CGG GAC GTG GCC TAC CTT CAG GCC CTT TCC CTG GAG CTT GCG 1987 Val Arg Arg Asp Val Ala Tyr Leu Gln Ala Leu Ser Leu Glu Leu Ala 455 460 465 GAG GAG ATC CGC CGC CTC GAG GAG GAG GAG TC TTC CGC TTG GCG GGC CAC 2035 Glu Glu Ile Arg Arg Leu Glu Glu Glu Val Phe Arg Leu Ala Gly His 470 475 480 480 CCC TTC AAC CTC AAC TCC CGG GAC CAG CTG GAA AGG GTG CTC TTT GAC 2083 Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp 485 490 495 GAG CTT AGG CTT CCC GCC TTG GGG AAG ACG CAA AAG ACA GGC AAG CGC 2131 Glu Leu Arg Leu Pro Ala Leu Gly Lys Thr Gln Lys Thr Gly Lys Arg 500 505 510 TCC ACC AGC GCC GCG GTG CTG GAG GCC CTA CGG GAG GCC CAC CCC ATC 2179 Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile 515 520 525 530 GTG GAG AAG ATC CTC CAG CAC CGG GAG CTC ACC AAG CTC AAG AAC ACC 2227 Val Glu Lys Ile Leu Gln His Arg Glu Leu Thr Lys Leu Lys Asn Thr 535 540 545 TAC GTG GAC CCC CTC CCA AGC CTC GTC CAC CCG AGG ACG GGC CGC CTC 2275 Tyr Val Asp Pro Leu Pro Ser Leu Val His Pro Arg Thr Gly Arg Leu 550 555 560 CAC ACC CGC TTC AAC CAG ACG GCC ACG GCC ACG GGG AGG CTT AGT AGC 2323 His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser 565 570 575 TCC GAC CCC AAC CT G CAG AAC ATC CCC GTC CGC ACC CCC TTG GGC CAG 2371 Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln 580 585 590 590 AGG ATC CGC CGG GCC TTC GTG GCC GAG GCG GGT TGG GCG TTG GTG GCC 2419 Arg Ile Arg Arg Ala Phe Val Ala Glu Ala Gly Trp Ala Leu Val Ala 595 600 605 610 CTG GAC TAT AGC CAG ATA GAG CTC CGC GTC CTC GCC CAC CTC TCC GGG 2467 615 620 625 GAC GAA AAC CTG ATC AGG GTC TTC CAG GAG GGG AAG GAC ATC CAC ACC 2515 Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Lys Asp Ile His Thr 630 635 640 CAG ACC GCA AGC TGG ATG TTC GGC GTC CCC CCG GAG GCC GTG GAC CCC 2563 Gln Thr Ala Ser Trp Met Phe Gly Val Pro Pro Glu Ala Val Asp Pro 645 650 655 CTG ATG CGC CGG GCG GCC AAG ACG GTG AAC TTC GGC GTC CTC TAC GGC 2611 Leu Met Arg Arg Ala Ala Lys Thr Val Asn Phe Gly Val Leu Tyr Gly 660 665 670 ATG TCC GCC CAT AGG CTC TCC CAG GAG CTT GCC ATC CCC TAC GAG GAG 2659 Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu 675 680 685 690 GCG GTG GCC TTT ATA GAG CGC TAC TTC CAA AGC TTC CCC AAG GTG CGG 2707 Ala Val Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg 695 700 705 GCC TGG ATA GAA AAG ACC CTG GAG GAG GGG AGG AAG CGG GGC TAC GTG 2755 Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Lys Arg Gly Tyr Val 710 715 720 GAA ACC CTC TTC GGA AGA AGG CGC TAC GTG CCC GAC CTC AAC GCC CGG 2803 Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Asn Ala Arg 725 730 735 GTG AAG AGC GTC AGG GAG GCC GCG GAG CGC ATG GCC TTC AAC ATG CCC 2851 Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro 740 745 750 GTC CAG GGC ACC GCC GCC GAC CTC ATG AAG CTC GCC ATG GTG AAG CTC 2899 Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu 755 760 765 770 Phe Pro Arg Leu Arg Glu Met Gly Ala Arg Met Leu Leu Gln Val His 775 780 785 GAC GAG CTC CTC CTG GAG GCC CCC CAA GCG CGG GCC GAG GAG GTG GCG 2995 Asp Glu Leu Leu Leu Glu Ala Pro Gln Ala Arg Ala Glu Glu Val Ala 790 795 800 GCT TTG GCC AAG GAG GCC ATG GAG AAG GCC TAT CCC CTC GCC GTG CCC 3043 Ala Leu Ala Lys Glu Ala Met Glu Lys Ala Tyr Pro Leu Ala Val Pro 805 810 815 CTG GAG GTG GAG GTG GGG ATG GGG GAG GAC TGG CTT TCC GCC AAG GGT 3091 Leu Glu Val Glu Val Gly Met Gly Glu Asp Trp Leu Ser Ala Lys Gly 820 825 830 TAGGGGGGCC CTGCTCTTCAGGAAGT CGCCTCCAGG 3151 GGAACGCCCT CTGGGCCTAC CAGGAGGCCT TTAGCCCCAA AGGTGCGGGT GAAGGCTTCC 3211 AGGCCCTGGG 3221
【図面の簡単な説明】[Brief description of drawings]
【図1】pUPL1の構築方法を示す図である。FIG. 1 is a diagram showing a method for constructing pUPL1.
フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 FI 技術表示箇所 C12R 1:01) Continuation of front page (51) Int.Cl. 5 Identification code Office reference number FI technical display area C12R 1:01)
Claims (5)
ermophirus)由来の耐熱性DNAポリメラーゼ又は該耐
熱性DNAポリメラーゼと機能的同等物をコードしてい
るDNA。1. Thermus th
ermophirus) -derived thermostable DNA polymerase or a DNA encoding a functional equivalent to the thermostable DNA polymerase.
た塩基配列又はそれらと均等な塩基配列をコードしてい
る請求項1に記載されたDNA。2. The DNA according to claim 1, wherein the DNA encodes the base sequence shown in SEQ ID NO: 1 of the sequence listing or a base sequence equivalent thereto.
ター。3. A recombinant vector containing the DNA of claim 1.
形質転換した形質転換体。4. A transformant obtained by transforming a host cell with the recombinant vector according to claim 3.
性DNAポリメラーゼを産生させ、ついで該耐熱性DN
Aポリメラーゼを採取することを特徴とする耐熱性DN
Aポリメラーゼの製造法。5. The transformant according to claim 4 is cultured to produce a thermostable DNA polymerase, and the thermostable DN is then produced.
Thermostable DN characterized by collecting A polymerase
Method for producing A polymerase.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP12464392A JPH05317058A (en) | 1992-05-18 | 1992-05-18 | Dna fragment having heat-resistant dna polymerase gene information and its use |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP12464392A JPH05317058A (en) | 1992-05-18 | 1992-05-18 | Dna fragment having heat-resistant dna polymerase gene information and its use |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH05317058A true JPH05317058A (en) | 1993-12-03 |
Family
ID=14890485
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP12464392A Pending JPH05317058A (en) | 1992-05-18 | 1992-05-18 | Dna fragment having heat-resistant dna polymerase gene information and its use |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH05317058A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110023493A (en) * | 2016-11-22 | 2019-07-16 | 东洋纺株式会社 | Taq DNA polymerase through changing |
-
1992
- 1992-05-18 JP JP12464392A patent/JPH05317058A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110023493A (en) * | 2016-11-22 | 2019-07-16 | 东洋纺株式会社 | Taq DNA polymerase through changing |
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