JPH0560356B2 - - Google Patents
Info
- Publication number
- JPH0560356B2 JPH0560356B2 JP62208332A JP20833287A JPH0560356B2 JP H0560356 B2 JPH0560356 B2 JP H0560356B2 JP 62208332 A JP62208332 A JP 62208332A JP 20833287 A JP20833287 A JP 20833287A JP H0560356 B2 JPH0560356 B2 JP H0560356B2
- Authority
- JP
- Japan
- Prior art keywords
- adenylate kinase
- ppak3
- plasmid
- ppak1
- pig
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 108020000543 Adenylate kinase Proteins 0.000 claims description 18
- 108020004705 Codon Proteins 0.000 claims description 10
- 229930182817 methionine Natural products 0.000 claims description 7
- 239000013604 expression vector Substances 0.000 claims description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 239000013598 vector Substances 0.000 claims description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- 102000002281 Adenylate kinase Human genes 0.000 claims 4
- 239000002773 nucleotide Substances 0.000 claims 3
- 125000003729 nucleotide group Chemical group 0.000 claims 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims 1
- 239000013612 plasmid Substances 0.000 description 17
- 102100032534 Adenosine kinase Human genes 0.000 description 14
- 238000000034 method Methods 0.000 description 9
- 239000002299 complementary DNA Substances 0.000 description 6
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- XTWYTFMLZFPYCI-KQYNXXCUSA-N 5'-adenylphosphoric acid Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O XTWYTFMLZFPYCI-KQYNXXCUSA-N 0.000 description 3
- XTWYTFMLZFPYCI-UHFFFAOYSA-N Adenosine diphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(O)=O)C(O)C1O XTWYTFMLZFPYCI-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 description 3
- 229950006790 adenosine phosphate Drugs 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000002703 mutagenesis Methods 0.000 description 3
- 231100000350 mutagenesis Toxicity 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 2
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- 108010077805 Bacterial Proteins Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 108010052418 (N-(2-((4-((2-((4-(9-acridinylamino)phenyl)amino)-2-oxoethyl)amino)-4-oxobutyl)amino)-1-(1H-imidazol-4-ylmethyl)-1-oxoethyl)-6-(((-2-aminoethyl)amino)methyl)-2-pyridinecarboxamidato) iron(1+) Proteins 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100029764 DNA-directed DNA/RNA polymerase mu Human genes 0.000 description 1
- 101710203526 Integrase Proteins 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- SVSKFMJQWMZCRD-MCDZGGTQSA-L MgADP Chemical compound [Mg+2].C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP(O)([O-])=O)[C@@H](O)[C@H]1O SVSKFMJQWMZCRD-MCDZGGTQSA-L 0.000 description 1
- 102000013009 Pyruvate Kinase Human genes 0.000 description 1
- 108020005115 Pyruvate Kinase Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 108010092809 exonuclease Bal 31 Proteins 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Description
〔産業上の利用分野〕
本発明は、ブタ−アデニレートキナーゼを微生
物等を用いて大量に生産するための発現ベクター
に関する。
このブタ−アデニレートキナーゼは、
Mg−ATP＋AMP⇔Mg−ADP＋ADP
の反応において触媒作用を行なう酸素で、アデノ
シン三リン酸（ATP）、アデニル酸（AMP）、ア
デノシン二リン酸（ADP）等の試薬の生産或い
はバイオリアクタ一等におけるATPすなわちエ
ネルギー再生用の酵素として有用なものである。
〔従来の技術〕
上記ブタ−アデニレートキナーゼは、ブタの筋
肉中に見い出される酵素で、このアミノ酸配列
は、次のものとして知られている〔エ、ヘイル他
（A.Heil、et.al.）、ヨーロピアン、ジヤーナル、
オブ、バイオケミストリー（European Journal
of Biochemistry）、43、p131（1974）〕。
[Industrial Application Field] The present invention relates to an expression vector for producing large amounts of porcine adenylate kinase using microorganisms and the like. This porcine adenylate kinase uses oxygen to catalyze the reaction of Mg-ATP + AMP ⇔ Mg-ADP + ADP, and uses reagents such as adenosine triphosphate (ATP), adenylic acid (AMP), and adenosine diphosphate (ADP). It is useful as an enzyme for the production of ATP or energy regeneration in bioreactors, etc. [Prior Art] The above-mentioned pig adenylate kinase is an enzyme found in pig muscle, and its amino acid sequence is known as the following [A. Heil, et.al. .), European, Journal,
Of, Biochemistry (European Journal
of Biochemistry), 43 , p131 (1974)].
本発明は、上記問題点を解決するための微生物
等を用いて、大量にしかも安価にブタ−アデニレ
ートキナーゼを生産させるための発現ベクターを
提供するものである。
〔問題点を解決するための手段〕
本発明者等は、ブタ−アデニレートキナーゼに
ついて鋭意研究を進めた結果、ブタ−アデニレー
トキナーゼのcDNAが次に示す塩基配列を有する
ことを知つた。
The present invention aims to solve the above-mentioned problems by providing an expression vector for producing pig adenylate kinase in large quantities and at low cost using microorganisms and the like. [Means for solving the problem] As a result of intensive research on pig adenylate kinase, the present inventors learned that the cDNA of pig adenylate kinase has the following base sequence. .
ブタ−アデニレートキナーゼcDNAの調製
ブタの筋肉組織をすりつぶし、グアニジウムイ
ソチオシアネートでタンパクを変性させ、塩化セ
シウム密度勾配遠心分離で、RNAを分離した。
次に、これをオリゴdTセルロースカラムを用い
て精製し、ポリアデニレートRNAを得た。この
ポリアデニレートRNAを鋳型として逆転写酵素
を用いて、cDNA−RNAの二本鎖を作り、この
うちRNAを大腸菌リボヌクレアーゼＨで分解し、
大腸菌DNAポリメラーゼにより、二本鎖の
cDNAを調製した。
上記で得たcDNAをターミナルトランスフエラ
ーゼで処理し、両3′末端にオリゴdCをつけ、プ
ラスミドpUC9（宝酒造(株)社製）をPstlで切断した
両末端に同様にしてオリゴdGをつけたものに、
挿入した。
このcDNAをサンガー法により分析した結果、
前記に示した塩基配列を有することが分つた。
発現ベクターの調製
（pPAK1）
上記プラスミドpUC9のPstIサイトにクローニ
ングしていたブタ−アデニレートキナーゼの
cDNAを制限酵素PstIで切り出し、両末端のＧ、
Ｃ部分をBal31ヌクレアーゼ除去した後、クレノ
ー酵素でブラントエンドにし、これを市販のプラ
スミドpKK223−３（フエルマシア社製、tacプロ
モーターを有するpBR322由来のプラスミド）の
SmaIサイトにつないだ。この改変プラスミドを
pPAK1とした。
この調製したプラスミドの塩基配列は、サンガ
ー法で確認した。
（pPAK1−２）
次に、上記pPAK1にホスフアイト法により合
成した5′−
CAGGAAACAGAATTCATGGAAGAGAAGC
ＴＧ−3′（AGGA部はSD配列、ATG部はアミノ末
端メチオニンコドン）をプライマーとして位置特
異的変異導入法により導入し、SD配列とアミノ
末端メチオニンコドン間の距離が10塩基のベクタ
ーpPAK1−２を調製した。
すなわち、第１図に示しように、pPAK1を制
限酵素PvuI及びScaIで切断したPtacサイト部と
同じpPAK1を制限酵素SmaIとBamHIで切断し
たAmprサイト部とに、上記合成プライマーを添
加し、100℃の温度で、３分間変性させ、30℃、
30分間保持して、再生し、次いで、４℃で、30分
間保持した。
次いで、上記プライマーを導入したプラスミド
を、DNAポリメラーゼ、４種のdNTP及び
T4DNAリガーゼで環状二本鎖とし、それを用い
て大腸菌を形質転換した。
目的物は、上記合成プライマーの放射性ラベリ
ングしたものをプローブとしてハイブリダイゼー
シヨン法でスクリーニングした。
この調製したプラスミドの塩基配列は、サンガ
ー法で確認した。このプラスミドをpPAK1−２
とした。
（pPAK3）
上記pPAK1−２にホスフアイト法により合成
した5′−AGAATTCATGATGGAAG−3′（ATG
部はアミノ末端メチオニンコドン）をプライマー
として位置特異的変異導入法により導入し、ベク
ターpPAK3を調製した。これは結果として、上
記ベクターpPAK1−２アイノ末端メチオニンコ
ドンの直前にATGを挿入したことになつている。
尚、この挿入したATGコドンとSD配列間は10塩
基である。
すなわち、第２図に示すように、pPAK1−２
を制限酵素PvuI及びScaIで切断したPtacサイト
部と同じpPAK1−２を制限酵素SmaIとBamHI
で切断したAmprサイト部とに、上記合成プライ
マーを添加し、100℃の温度で、３分間変性させ、
30℃、30分間保持して、再生し、次いで、４℃
で、30分間保持した。
次いで、上記プライマーを導入したプラスミド
を、DNAポリメラーゼ、４種をdNTP及び
T4DNAリガーゼで環状二本鎖とし、それを用い
て、大腸菌を形質転換した。
目的物は、上記合成プライマーの放射性ラベリ
ングしたものをプローブとしてハイブリダイゼー
シヨン法でスクリーニングした。
この調製したプラスミドの塩基配列は、サンガ
ー法で確認した。このプラスミドをpPAK3とし
た。
（pPAK3−１〜３−３）
上記プラスミドpPAK1−２及びpPAK3の調製
と同様と操作で、pPAK3のSD配列と挿入した
ATGコドン間の塩基配列をAGAATTC、
AAGAATTC、AATTCAGAATTC、と７塩
基、８塩基、12塩基に変え、それぞれpPAK3−
１、pPAK3−２及びpPAK3−３とした。これら
の調製したプラスミドの塩基配列は、サンガー法
で確認した。
ブタ−アデニレートキナーゼの発現
上記で調製したプラスミドを大腸菌E.coli
JM109にそれぞれ導入し、これを50μｇ／mlのア
ンピシリンを含むLB培地〔バクトトリプトン
（Bacto−tryptone）10ｇ／、バクトイースト
エキストラクト（Bacto−yeast extract）５
ｇ／、NaCl10ｇ／、PH7.5〕で培養し、１ｍ
Ｍのイソプロピルチオガラクトシド（IPTG）に
より誘導した。一晩培養後、遠心分離で菌体を集
め、ソニケータで菌体を壊した後、遠心分離で上
清を集め、さらに回転数25000rpmの超遠心分離
機で１時間遠心分離し、その上清をとつた。この
上清について、エンザイムリンクドイムノソルベ
ントアツセイ（ELISA）とピルベートキナーゼ
とラクテートデヒドロゲナーゼを組み合わせ、還
元型ニコチンアミドアデニンジヌクレオチドの
340nｍ吸収の減少を測定した活性を求める酵素
アツセイによりブタ−アデニレートキナーゼの同
定を行つた。
一方、発現量の比較は、遠心分離で集めた菌体
を直接可溶化し、SDS−ポリアクリルアミドゲル
電気泳動を行い、クマシーブルーで染色後、デン
シトメーターでタンパクバンドの強度を測定する
ことにより行つた。
この結果、pPAK1を導入したものは、ほとん
ど発現せず、pPAK1−２のものは、菌体全タン
パクの１％、pPAK3では、菌体全タンパクの70
％、pPA3−１、pPAK3−２及びpPAK3−３で
は、それぞれ55％、55％、35％であつた。
そして、pPAK3が挿入された菌株はE.coli
JM 109 pPAK3、FERM BP−1452として微工
研に寄託されている。
このように、ブタ−アデニレートキナーゼのア
ミノ末端メチオニンコドンの直前にATGの塩基
配列を挿入することによりブタ−アデニレートキ
ナーゼの生産量が著しく高まることが分かる。さ
らに、SD配列と挿入したATGコドン間の距離は
10塩基がより好ましいことも分かつた。
〔発明の効果〕
本発明は、発現ベクターにブタ−アデニレート
キナーゼのアミノ酸配列をコードする塩基と当該
塩基配列のアミノ末端メチオニンコドンの直前に
ATGの塩基配列とを含ませたため、大量にしか
も安価にブタ−アデニレートキナーゼを生産する
ことができるという格別の効果を奏するものであ
る。
Preparation of Pig Adenylate Kinase cDNA Pig muscle tissue was ground, proteins were denatured with guanidinium isothiocyanate, and RNA was isolated by cesium chloride density gradient centrifugation.
Next, this was purified using an oligo dT cellulose column to obtain polyadenylate RNA. This polyadenylate RNA is used as a template and reverse transcriptase is used to create a cDNA-RNA double strand, of which RNA is degraded with E. coli ribonuclease H.
Double-stranded DNA polymerase
cDNA was prepared. The cDNA obtained above was treated with terminal transferase and oligo dC was added to both 3' ends, and oligo dG was added to both ends of plasmid pUC9 (manufactured by Takara Shuzo Co., Ltd.) which had been cut with Pstl. To things,
Inserted. As a result of analyzing this cDNA by Sanger method,
It was found that it had the base sequence shown above. Preparation of expression vector (pPAK1) Porcine adenylate kinase cloned into the PstI site of the above plasmid pUC9.
Cut out the cDNA with the restriction enzyme PstI, and
After removing the C portion with Bal31 nuclease, it was made blunt-ended with Klenow enzyme, and this was transformed into a commercially available plasmid pKK223-3 (manufactured by Fermacia, a pBR322-derived plasmid with a tac promoter).
Connected to SmaI site. This modified plasmid
It was named pPAK1. The base sequence of this prepared plasmid was confirmed by Sanger method. (pPAK1-2) Next, 5′-
CAGGAAACAGAATTCATGGAAGAGAAGC
Vector pPAK1-2, in which the distance between the SD sequence and the amino-terminal methionine codon is 10 bases, was introduced by position-directed mutagenesis using TG-3' (AGGA part is SD sequence, ATG part is amino-terminal methionine codon) as a primer. was prepared. That is, as shown in FIG. 1, the above synthetic primer was added to the Ptac site obtained by cutting pPAK1 with the restriction enzymes PvuI and ScaI and the Amp r site obtained by cutting the same pPAK1 with the restriction enzymes SmaI and BamHI. Denature for 3 minutes at a temperature of 30°C,
Hold for 30 minutes to regenerate, then hold at 4°C for 30 minutes. Next, the plasmid into which the above primers were introduced was treated with DNA polymerase, four types of dNTPs and
This was made into a circular double strand using T4 DNA ligase, and used to transform E. coli. The target product was screened by a hybridization method using the radiolabeled synthetic primer described above as a probe. The base sequence of this prepared plasmid was confirmed by Sanger method. This plasmid is pPAK1-2
And so. (pPAK3) 5′-AGAATTCATGATGGAAG-3′ (ATG
The vector pPAK3 was prepared by position-directed mutagenesis using the amino-terminal methionine codon as a primer. This resulted in the insertion of ATG immediately before the aino-terminal methionine codon of the vector pPAK1-2.
Note that there are 10 bases between the inserted ATG codon and the SD sequence. That is, as shown in Figure 2, pPAK1-2
pPAK1-2, which is the same as the Ptac site cut with restriction enzymes PvuI and ScaI, was cut with restriction enzymes SmaI and BamHI.
The above synthetic primer was added to the Amp r site section cut with , and denatured at a temperature of 100°C for 3 minutes.
Hold at 30°C for 30 minutes to regenerate, then 4°C
and held for 30 minutes. Next, the plasmid containing the above primers was treated with DNA polymerase, dNTP and
This was made into a circular double strand using T4 DNA ligase, and used to transform Escherichia coli. The target product was screened by a hybridization method using the radiolabeled synthetic primer described above as a probe. The base sequence of this prepared plasmid was confirmed by Sanger method. This plasmid was named pPAK3. (pPAK3-1 to 3-3) Insert the SD sequence of pPAK3 using the same procedure as for the preparation of plasmids pPAK1-2 and pPAK3 above.
The base sequence between the ATG codons is AGAATTC,
Changed to AAGAATTC, AATTCAGAATTC, 7 bases, 8 bases, 12 bases, respectively, pPAK3-
1, pPAK3-2 and pPAK3-3. The base sequences of these prepared plasmids were confirmed by Sanger method. Expression of pig adenylate kinase The plasmid prepared above was added to E. coli.
JM109 and LB medium containing 50 μg/ml ampicillin [Bacto-tryptone 10 g/, Bacto-yeast extract 5
g/, NaCl 10 g/, PH 7.5] and 1 m
It was induced by isopropylthiogalactoside (IPTG) of M. After culturing overnight, the bacterial cells were collected by centrifugation, broken with a sonicator, the supernatant was collected by centrifugation, and the supernatant was further centrifuged for 1 hour in an ultracentrifuge at 25,000 rpm. Totsuta. This supernatant was analyzed using an enzyme-linked immunosorbent assay (ELISA) in combination with pyruvate kinase and lactate dehydrogenase to detect reduced nicotinamide adenine dinucleotide.
Porcine adenylate kinase was identified by an enzyme assay for activity determined by a decrease in absorption at 340 nm. On the other hand, expression levels can be compared by directly solubilizing bacterial cells collected by centrifugation, performing SDS-polyacrylamide gel electrophoresis, staining with Coomassie blue, and measuring the intensity of protein bands with a densitometer. I went. As a result, the cells into which pPAK1 was introduced showed almost no expression, those with pPAK1-2 expressed only 1% of the total bacterial protein, and those with pPAK3 expressed 70% of the total bacterial protein.
%, pPA3-1, pPAK3-2, and pPAK3-3 were 55%, 55%, and 35%, respectively. The strain into which pPAK3 was inserted is E. coli.
JM 109 pPAK3, deposited with the Institute of Fine Technology as FERM BP-1452. Thus, it can be seen that the production of pig adenylate kinase is significantly increased by inserting the ATG base sequence immediately before the amino terminal methionine codon of pig adenylate kinase. Furthermore, the distance between the SD sequence and the inserted ATG codon is
It was also found that 10 bases is more preferable. [Effects of the Invention] The present invention provides an expression vector containing a base encoding the amino acid sequence of porcine adenylate kinase and a base immediately before the amino terminal methionine codon of the base sequence.
Since it contains the base sequence of ATG, it has the special effect of being able to produce pig adenylate kinase in large quantities and at low cost.
第１図は、プラスミドpPAK1−２を、また第
２図は、プラスミドpPAK3を位置特異的変異導
入法により調製する過程の概略を示した図であ
る。
FIG. 1 is a diagram schematically showing the process of preparing plasmid pPAK1-2 and FIG. 2 is plasmid pPAK3 by position-directed mutagenesis.
Claims (1)
をコードする塩基配列と当該塩基配列のアミノ末
端のメチオニンコドンの直前のATGの塩基配列
とを含んでなるブタ−アデニレートキナーゼ発現
ベクター。 ２ ブタ−アデニレートキナーゼのアミノ酸配列
をコードする塩基配列が、 【表】 【表】 で表されることを特徴とする特許請求の範囲第１
項記載のブタ−アデニレートキナーゼ発現ベクタ
ー。[Scope of Claims] 1. Porcine adenylate kinase expression comprising a nucleotide sequence encoding the amino acid sequence of porcine adenylate kinase and an ATG nucleotide sequence immediately before the methionine codon at the amino terminus of the nucleotide sequence. vector. 2 Claim 1, characterized in that the base sequence encoding the amino acid sequence of porcine adenylate kinase is represented by [Table] [Table]
Porcine adenylate kinase expression vector as described in .
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20833287A JPS6451087A (en) | 1987-08-24 | 1987-08-24 | Swine adenylate kinase manifestation vector |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP20833287A JPS6451087A (en) | 1987-08-24 | 1987-08-24 | Swine adenylate kinase manifestation vector |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6451087A JPS6451087A (en) | 1989-02-27 |
| JPH0560356B2 true JPH0560356B2 (en) | 1993-09-02 |
Family
ID=16554515
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP20833287A Granted JPS6451087A (en) | 1987-08-24 | 1987-08-24 | Swine adenylate kinase manifestation vector |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6451087A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2747379B2 (en) * | 1991-05-31 | 1998-05-06 | 昭和アルミニウム株式会社 | Heat exchanger |
| JP3566045B2 (en) * | 1997-10-02 | 2004-09-15 | アルプス電気株式会社 | Device for mounting recording media |
-
1987
- 1987-08-24 JP JP20833287A patent/JPS6451087A/en active Granted
Non-Patent Citations (1)
| Title |
|---|
| EUROPIAN JOURNAL OF BIOCHEMISTRY=1974 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6451087A (en) | 1989-02-27 |
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