JPS63102695A - Production of photoprotein aequorin using extracellular secretory vector of escherichia coli - Google Patents
Production of photoprotein aequorin using extracellular secretory vector of escherichia coliInfo
- Publication number
- JPS63102695A JPS63102695A JP24909886A JP24909886A JPS63102695A JP S63102695 A JPS63102695 A JP S63102695A JP 24909886 A JP24909886 A JP 24909886A JP 24909886 A JP24909886 A JP 24909886A JP S63102695 A JPS63102695 A JP S63102695A
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- aequorin
- coli
- escherichia coli
- vector
- protein
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Links
- 108010000239 Aequorin Proteins 0.000 title claims abstract description 42
- 241000588724 Escherichia coli Species 0.000 title claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 title claims description 11
- 239000013598 vector Substances 0.000 title abstract description 8
- 230000003248 secreting effect Effects 0.000 title abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 23
- 239000013612 plasmid Substances 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 238000010367 cloning Methods 0.000 claims abstract description 5
- 230000028327 secretion Effects 0.000 claims abstract description 5
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 12
- 230000001580 bacterial effect Effects 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 11
- 239000013606 secretion vector Substances 0.000 claims description 8
- 241000242583 Scyphozoa Species 0.000 claims description 6
- 239000013604 expression vector Substances 0.000 claims description 5
- 101150115693 ompA gene Proteins 0.000 claims description 5
- 101100295756 Acinetobacter baumannii (strain ATCC 19606 / DSM 30007 / JCM 6841 / CCUG 19606 / CIP 70.34 / NBRC 109757 / NCIMB 12457 / NCTC 12156 / 81) omp38 gene Proteins 0.000 claims description 4
- 101150042295 arfA gene Proteins 0.000 claims description 4
- 101150087557 omcB gene Proteins 0.000 claims description 4
- 108010079246 OMPA outer membrane proteins Proteins 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 3
- 229940116108 lactase Drugs 0.000 claims description 2
- 239000002299 complementary DNA Substances 0.000 abstract description 9
- 102000004895 Lipoproteins Human genes 0.000 abstract 1
- 108090001030 Lipoproteins Proteins 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 20
- 150000002500 ions Chemical class 0.000 description 8
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 description 5
- 239000012634 fragment Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 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 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Tropical Medicine & Parasitology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔技術の分野〕
本発明は発光蛋白エクオリンの遺伝子を用いてのエクオ
リン活性を有する蛋白質の製造法に関する。更に詳しく
は、大腸菌の菌体外分泌ベクターを用いて、大腸菌菌体
外へエクオリン活性を有する蛋白を分泌製造する方法に
関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Technology] The present invention relates to a method for producing a protein having aequorin activity using a gene for the photoprotein aequorin. More specifically, the present invention relates to a method for secreting and producing a protein having aequorin activity outside the E. coli cell using an E. coli extracellular secretion vector.
エクオリンは、米国ワシントン州フライデーハーバ−島
近郊に生息する発光クラゲより分はされたカルシウム受
容発光蛋白質である。Aequorin is a calcium-accepting photoprotein isolated from the luminescent jellyfish that lives near Friday Harbor Island, Washington, USA.
エクオリン１分子に２分子（あるいは３分子）のＣａ２
”イオンが結合することによりエクオリン分子中に含
まれる活性化状態にあるセレンテラジンが酸化され、光
と ＣＯ２を放出する。この発光には、Ｃａ２°イオン
が不可欠なことより、エクオリンを用いてＣａ２・を特
異的に定量検出すること（が可能である。 Ｃａ２°イ
オンの検出限界は１０−９Ｍ程度である。One molecule of aequorin contains two (or three) molecules of Ca2.
``When the ions combine, coelenterazine in the activated state contained in the aequorin molecule is oxidized and releases light and CO2.Since Ca2° ions are essential for this light emission, aequorin is used to oxidize the activated coelenterazine. It is possible to specifically and quantitatively detect Ca2° ions. The detection limit for Ca2° ions is about 10-9M.
現在までに、細胞膜の興奮、筋収縮、細胞の分泌活動、
伝達物質の遊離、ホルモンの作用若しくは細胞間の連結
、融合などの多くの細胞機能の発現に細胞内Ｃａ２・イ
オンが重要な役割を果していることが、広く認められて
いる。これらの細胞機能と細胞内Ｃａ２ ’イオンとの
関係を明らかにするためには、細胞内Ｃａ２・濃度の経
時的定量が必要となってくる。細胞内Ｃａ２ ”イオン
の相対的、経時的変化の測定には、実験系が比較的単純
でＳＮ比のよいＣａ２ ’イオンのシグナルが記録でき
るためエクオリンが用いられる。To date, cell membrane excitation, muscle contraction, cell secretory activity,
It is widely accepted that intracellular Ca2/ions play an important role in the expression of many cellular functions such as the release of transmitters, the action of hormones, and the connections and fusion between cells. In order to clarify the relationship between these cellular functions and intracellular Ca2' ions, it is necessary to quantify the intracellular Ca2 concentration over time. Aequorin is used to measure relative changes in intracellular Ca2' ions over time because the experimental system is relatively simple and signals of Ca2' ions can be recorded with a good signal-to-noise ratio.
しかしながら、エクオリンの天然からの分離は発光クラ
ゲ１０トンから２００ｍｇ程度にすぎず、生産量は十分
でなく、一定の供給が保証されていない。However, the amount of aequorin isolated from nature is only about 200 mg from 10 tons of luminescent jellyfish, and the production amount is not sufficient and a constant supply is not guaranteed.
本発明者らは、前述の問題を解決すべく、研究を行い、
遺伝子工学の手法を用い、クラゲ発光蛋白エクオリンの
ｃＤＮＡクローンを取得し、このｃＤＮＡクローンプラ
スミドをｐＡＱ４４０と名付けた（特願昭５９−１７８
１２５号）０次いで、この遺伝子（ｐＡＱ４４０）を大
腸菌の発現プロモーターを有するプラスミドに挿入し、
トランスフォーメーションした菌体を用い、天然型及び
融合型の発光蛋白エクオリンを大腸菌体内で、俺率的に
生産することが出きた（＃願昭８０−２８０２５９号）
。The present inventors conducted research in order to solve the above-mentioned problem,
Using genetic engineering techniques, we obtained a cDNA clone of the jellyfish photoprotein aequorin, and named this cDNA clone plasmid pAQ440 (Japanese Patent Application No. 59-178).
No. 125) 0 Next, this gene (pAQ440) was inserted into a plasmid having an E. coli expression promoter,
Using transformed bacterial cells, we were able to produce naturally occurring and fused photoprotein aequorin in Escherichia coli cells (#Grant No. 80-280259)
.
しかしながら、該エクオリン蛋白の安定生産の為には、
大腸菌の菌体外に該蛋白を分泌せしめるような発現ベク
ターが要望される。However, for stable production of the aequorin protein,
There is a need for an expression vector that allows the protein to be secreted outside the E. coli cells.
木発明者らは、上述の要望を満足すべく研究の結果、上
述のｐＡＱ４４０を用い、大腸菌の外膜蛋白Ａ分泌用遺
伝子及びリボ蛋白（ｌｐｐ）のプロモーターと接続する
ことにより、エクオリン活性を有するエクオリン蛋白を
大腸菌の菌体外に放出する分泌型発現ベクターを構築し
、該ベクターを利用することにより、大腸菌の菌体内で
生産されたエクオリン蛋白を大腸菌４体外に爺率的に放
出し生産させることに成功した。As a result of research to satisfy the above-mentioned needs, the inventors used the above-mentioned pAQ440 and connected it to the E. coli outer membrane protein A secretion gene and the promoter of riboprotein (lpp), thereby creating a protein with aequorin activity. By constructing a secretory expression vector that releases aequorin protein outside the E. coli cells, and using this vector, the aequorin protein produced within the E. coli cells is efficiently released and produced outside the E. coli cells. It was very successful.
以上の記述から明らかなように本発明の目的は、該分泌
型発現ベクターならびに該ベクターにクローニングして
得られたプラスミドを大腸菌にトランスホーメートする
ことにより、エクオリン蛋白の菌体外分泌による製造法
を提供することである。As is clear from the above description, the purpose of the present invention is to develop a method for producing aequorin protein by extracellular secretion by transforming E. coli with the secretory expression vector and the plasmid obtained by cloning into the vector. It is to provide.
本発明は、下記（１）の主要構成と（２）および（３）
の実施態様的構成を有する。The present invention has the following main configuration (1) and (2) and (3)
It has an embodiment configuration.
（１）クラゲ発光蛋白エクオリンの生合成を特定する遺
伝子（ｐＡＱ４４０）を用いて菌体外分泌ベクターにク
ローニングして得られたプラスミドを大腸菌にトランス
ホーメートすることによりエクオリン活性な有する蛋白
質を該大腸菌の菌体外に分泌させることを特徴とする大
腸菌の菌体外分泌ベクターを用いる発光蛋白エクオリン
の製造法。(1) Using the gene (pAQ440) that specifies the biosynthesis of the jellyfish photoprotein aequorin, the resulting plasmid is cloned into an extracellular secretion vector, and the resulting plasmid is transformed into E. coli to transform the protein with aequorin activity into the E. coli. A method for producing the photoprotein aequorin using an extracellular secretion vector of Escherichia coli, which is characterized in that it is secreted outside the bacterial cell.
（２）大腸菌のリボ蛋白（１ｐｐ）のプロモーターの下
流に大腸菌の外膜蛋白Ａ（ｏｍｐＡ）の分泌の為のシグ
ナルペプチドをコードする遺伝子及びエクオリン遺伝子
（ｐＡＱ４４０）を接続したプラスミドを用いる前記第
（１）項に記載の製造法。(2) Using a plasmid in which a gene encoding a signal peptide for the secretion of E. coli outer membrane protein A (ompA) and the aequorin gene (pAQ440) are connected downstream of the E. coli riboprotein (1pp) promoter. The manufacturing method described in section 1).
（３）シグナルペプチドをコードする遺伝子として大腸
菌のβ−ラクターゼのシグナルペプチドを有する発現ベ
クターを用いる前記第（１）項に記載の方法。(3) The method according to item (1) above, which uses an expression vector having a signal peptide of β-lactase of E. coli as a gene encoding the signal peptide.
本発明の構成と効果につき以下に詳述する。The configuration and effects of the present invention will be explained in detail below.
先づ、クラゲ発光蛋白エクオリンの生合成を特定する遺
伝子（ｐＡＱ４４０）の取得については、上述のように
本発明者らによる特願昭５９−１７８１２５号（特開昭
８ｌ−１３５５Ｈ号）に詳述されている。First, the acquisition of the gene (pAQ440) that specifies the biosynthesis of the jellyfish photoprotein aequorin is described in detail in Japanese Patent Application No. 178125-1982 (Japanese Patent Application Laid-open No. 81-1355H) by the present inventors, as mentioned above. has been done.
このｐＡＱ４４０を用いる菌体外分泌ベクターの構築法
については後述の実施例１（イ）に例示されているよう
に、例えば、高コピーのクローニングベクターｐＵｃ
８の…Ｒ−ＨｉｎｄＩ［［部分をそれぞれの制限酵素で
消化後、その部分にｃＤＮＡクローンｐＡＱ４４０Ｊ：
り得られたエクオリンｃＤＮＡ…Ｒ−Ｈｉｎｄ ｍフラ
グメントをサブクローニングすることによりｐｉＱＨＥ
を取得する。Regarding the method of constructing an extracellular secretion vector using this pAQ440, as illustrated in Example 1 (a) below, for example, a high copy cloning vector pUc
After digesting the...R-HindI [[ part of 8 with each restriction enzyme, cDNA clone pAQ440J was added to that part:
piQHE by subcloning the aequorin cDNA...R-Hind m fragment obtained.
get.
次に、該ベクターへのクローニングによるプラスミドの
取得については後述の実施例１（ｏ）に例示さレテイル
ヨうに、ｐｉＱ ８ ＨＥ（７） Ｓｃａ Ｉ −Ｈｉ
ｎｄｍ消化後、その部分にｐＩＮ−ｍｏ閣ｐＡＩよりＩ
ＰＰプロモーター及びｏｍｐＡ遺伝子を含む５ｃａｌ
−Ｈｉｎｄｍフラグメントを挿入することにより、ｐｉ
ＰＨＥを取得する。Next, the acquisition of a plasmid by cloning into the vector is exemplified in Example 1(o) below.
After ndm digestion, I was added to that part from pIN-mo pAI.
5cal containing PP promoter and ompA gene
-By inserting the Hindm fragment, pi
Get PHE.
該プラスミドの大腸菌へのトランスホーメーション方法
は、公知方法に従う、該大腸菌を用いてのエクオリン蛋
白の生産方法は、後述の実施例２に例示されているよう
に、該大腸菌を所定の培養液中で培養し、該培養液に発
現誘導試薬を加えて所定の該試薬濃度とし更に所定条件
（温度、時間）で培養した培養物を遠心集菌し洗浄する
。The method for transforming the plasmid into E. coli is according to a known method. The method for producing aequorin protein using the E. coli is as exemplified in Example 2 below. An expression-inducing reagent is added to the culture solution to obtain a predetermined concentration of the reagent, and the culture is cultured under predetermined conditions (temperature, time) by centrifugation and washing.
かくして得られた菌体から、公知方法（Ｎｅｉｅｔ ａ
ｌ、Ｊ、Ｂｉｏｌ Ｃｈｅｌｌ、 ２４０巻３８８５−
３ｉ３９２（１９８５） ）に従ってスフェロプラスト
を作成する。From the bacterial cells thus obtained, a known method (Neiet a
l, J, Biol Chell, vol. 240, 3885-
3i392 (1985)).
このスフェロプラストを公知方法で破壊し、菌体、スフ
ェロプラストならびに処理剤菌体の夫々についてアクア
リンの酵素活性を測定する。The spheroplasts are destroyed by a known method, and the enzyme activity of aquarin is measured for each of the bacterial cells, spheroplasts, and treatment agent bacteria.
後述実施例２の表１にも示されているように菌体のスフ
ェロプラスト以外に処理菌体上清に該活性の大部分（例
えば９５％以上）が見出されることにより、エクオリン
が菌体外に分泌されていることが明らかである。As shown in Table 1 of Example 2 below, most of the activity (for example, 95% or more) is found in the treated bacterial cell supernatant other than in the spheroplasts of bacterial cells. It is clear that it is secreted externally.
以下実施例によって本発明を説明する。The present invention will be explained below with reference to Examples.
実施例１：ｌｐｐプロモーター及びｏｍｐＡのシグナル
ペプチドをコードする遺伝子へのエクオリンｃＤＮＡ遺
伝子の挿入（図１−ａ）（イ）ｐｉＱ８ＨＨの作成
高コピーのクローニングベクターｐＵｃ ８のＥｃｏＲ
Ｉ −Ｈｉｎｄｍ部分を、それぞれの制限酵素で消化後
その部分にｃＤＮＡクローンｐＡＱ４４０より得られた
エクオリンｃＤＮＡのＥｃｏＲＩ −）１ｉｎｄｍフラ
グメントを、サブクローニングを行うことにより、ｐｉ
ＱＨＥを作成した。Example 1: Insertion of the aequorin cDNA gene into the lpp promoter and the gene encoding the signal peptide of ompA (Figure 1-a) (a) Creation of piQ8HH EcoR of high copy cloning vector pUc 8
After digesting the I-Hindm portion with the respective restriction enzymes, the EcoRI-)1indm fragment of the aequorin cDNA obtained from the cDNA clone pAQ440 was subcloned into that portion.
Created QHE.
（ａ）ｐｉＰＨＨの作成
ｐｉＱ ８ＨＥの謹Ｉ−…ｄｍ消化後、その部分にｐｌ
Ｎ −ｍｏｍｐＡＩよりＩｐＰプロモーター及びｏ＋＊
ｐＡ遺伝子な含む５ｅａｌ −Ｈｉｎｄｍフラグメント
を挿入しｐｉＰＨＥを作成した。(a) Creation of piPHH piQ 8HE's I-...After dm is digested, pl into that part.
IpP promoter and o+* from N-mompAI
A 5eal-Hindm fragment containing the pA gene was inserted to create piPHE.
上記の方法で作成したｐｉＰＨＥは、シグナルペプチド
を有するエクオリン蛋白を生産することができる０図１
−ｂに、シグナルペプチドとエクオリン遺伝子の構造示
す。piPHE created by the above method can produce aequorin protein with a signal peptide.
-b shows the structure of the signal peptide and the aequorin gene.
実施例２：大腸菌を用いてのエクオリンの生産方法
前述の発現分泌ベクターにクローニングして作成したプ
ラスミドｐ １Ｐ）ＩＥを３．■１（ＪＡ２２１）株に
トランスフォーメイションを行いエクオリン活性を有す
る蛋白を生産させた。Example 2: Method for producing aequorin using E. coli Plasmid p1P) IE, which was created by cloning into the expression secretion vector described above, was used in 3. 1 (JA221) strain was transformed to produce a protein having aequorin activity.
すなわち、１００層８／諺文度のアンビミリンを含むＬ
Ｂｂｒｏｔｈ １０ｍ文に１／１００ ｉ１２時間培
養の上記菌体（プラスミドｐｉＰＨＥを含む）を添加後
３７℃２時間培養し、該培養液に発現誘導試薬ＩＰＴＧ
（ｉｓｏｐｒｏｐｙｌ−β−ｔｈｉｏｇａｌｏｉｔｏｐ
ｙｒａｎｏｓｉｏｌｅ）を最絆濃度１ｍＭになるよう添
加し、さらに３７℃で４時間培養した。That is, L containing 100 layer 8/proverbial ambimirin
The above bacterial cells (containing plasmid piPHE) cultured at 1/100 i for 12 hours were added to Bbroth 10m culture, and cultured at 37°C for 2 hours, and the expression induction reagent IPTG was added to the culture solution.
(isopropyl-β-thiogaloitop
yranosiole) was added to give a maximum concentration of 1 mM, and the cells were further cultured at 37°C for 4 hours.
以上のようにして得られた培養物を５００Ｏｒｐｍで１
０分間（Ｈ４ｔａｃｈｉ ＲＰ２００−ター）遠心集菌
後、Ｍ８培地で洗浄する。The culture obtained as above was heated at 500 rpm for 1
After centrifuging for 0 minutes (H4tachi RP200-ter) to collect bacteria, wash with M8 medium.
そしてＮｅｉ らの方法（Ｊ、 Ｂｉｏｌ、 Ｃｈｅｌ
ｌ、 ２４０巻３６８５−３Ｈ２（１ｆｌｌＥｉ５）
）に従って、菌体からスフェロプラストを作成した。す
なわち、冷０．０３Ｍ Ｔｒｉｓ−ＨＣＩ（ｐＨ８，１
）で２度洗浄後、２０％５ｕｃｒａｓｅ−０，０３ＭＴ
ｒｉｓ−ＨＣＩ（ｐＨ８，１）、１ｍＭＥＤＴＡを含む
溶液１０ｍ１を加え、１０分間ゆっくりと振とう後１３
，００Ｏｒｐｍで１０分間遠心後、沈殿に冷水１０膳文
を加え１０分間ゆっくり振とう後、同様に遠心後沈殿に
スフェロプラストが得られる。このものに、１．５＋ｓ
文のＴｒｉｓ−ＨＣＩｐ）ｌ ７．８．１０ｍＭＥＤＴ
Ａにとかしスフェロプラストは超音波処理で破壊し、ア
クアリンの酵素活性を測定した。and the method of Nei et al. (J, Biol, Chel
l, Volume 240 3685-3H2 (1fllEi5)
) Spheroplasts were created from the bacterial cells. That is, cold 0.03M Tris-HCI (pH 8.1
) After washing twice with 20% 5ucrase-0,03MT
Add 10 ml of a solution containing ris-HCI (pH 8,1) and 1 mM EDTA, and shake slowly for 10 minutes.
After centrifugation for 10 minutes at ,000 rpm, 10 servings of cold water were added to the precipitate, and after shaking slowly for 10 minutes, spheroplasts were obtained in the precipitate after centrifugation in the same manner. For this one, 1.5+s
Sentence Tris-HCIp)l 7.8.10mMEDT
The spheroplasts dissolved in A were destroyed by sonication, and the enzymatic activity of aquarin was measured.
その結果を表１に示す、明らかに、菌体のスフェロプラ
スト以外（処理菌体上清）にその活性の９５％以上が検
出され、エクオリンを菌体外にて産生することが可能と
なった。The results are shown in Table 1. Clearly, more than 95% of the activity was detected in cells other than bacterial spheroplasts (treated bacterial cell supernatant), indicating that it was possible to produce aequorin outside the bacterial cells. Ta.
（表１） 酵、素話性の測定
註０表で、ｒ、１．ｕ、は相対発光値であり、１ ｒ、
ｌｕ、＝ １．９Ｘ １０３ベクトルに相当する。(Table 1) In the measurement note 0 table of enzyme and speech, r, 1. u, is the relative luminescence value, 1 r,
lu, = 1.9X corresponds to 103 vectors.
測定はラボサイエンス社のルミノフォトメーター（ＴＯ
４０００）を用い、基質セレンテラジン０．２ｎｇ。Measurement was carried out using a Luminophotometer (TO) manufactured by LabScience.
4000) and 0.2 ng of the substrate coelenterazine.
５鉢文の２−メルカプトエタノールを酵素粗抽出液（１
００ｇＪｌ）に添加し、３０ｍＭ ＣａＣｌ２を １０
０ｐｕ注入し、測定した。5. 2-mercaptoethanol was added to the crude enzyme extract (1
00gJl) and 30mM CaCl2
0pu was injected and measured.
図１ （ａ）は、本発明の菌体外分泌ベクターへのエク
オリン遺伝子の挿入方法を説ＩＩ したちのである。
同図において、
ＩｐＰＰ：リボプロティンのプロモータ一部分１ａｃＰ
ｏ：β−ガラクトミダーゼのプロモータ一部分とオペレ
ータ一部分
７ ：ｏｍｐＡのシグナルペプチドの遺伝子部分を示
す。
図１（ｂ）は、本発明に係るシグナルペプチドとエクオ
リン遺伝子の構造を説明したものである。
同図において、
はシグナルペプチド（２１アミノ酸）を、以上
図１FIG. 1(a) shows the method for inserting the aequorin gene into the extracellular secretion vector of the present invention. In the same figure, IpPP: riboprotein promoter part 1acP
o: Promoter part and operator part of β-galactomidase 7: Shows the gene part of the signal peptide of ompA. FIG. 1(b) illustrates the structure of the signal peptide and aequorin gene according to the present invention. In the same figure, indicates the signal peptide (21 amino acids);
Claims (3)
伝子（ｐＡＱ４４０）を用いて菌体外分泌ベクターにク
ローニングして得られたプラスミドを大腸菌にトランス
ホーメートすることによりエクオリン活性を有する蛋白
質を該大腸菌の菌体外に分泌させることを特徴とする大
腸菌の菌体外分泌ベクターを用いる発光蛋白エクオリン
の製造法。(1) Using the gene (pAQ440) that specifies the biosynthesis of the jellyfish photoprotein aequorin, the plasmid obtained by cloning into an extracellular secretion vector is transformed into E. coli, and the protein with aequorin activity is transformed into the E. coli. A method for producing the photoprotein aequorin using an extracellular secretion vector of Escherichia coli, which is characterized in that it is secreted outside the bacterial cell.
流に大腸菌の外膜蛋白Ａ（ｏｍｐＡ）の分泌の為のシグ
ナルペプチドをコードする遺伝子及びエクオリン遺伝子
（ｐＡＱ４４０）を接続したプラスミドを用いる特許請
求の範囲第（１）項に記載の製造法。(2) A patent claim using a plasmid in which a gene encoding a signal peptide for the secretion of E. coli outer membrane protein A (ompA) and the aequorin gene (pAQ440) are connected downstream of the E. coli riboprotein (lpp) promoter. The manufacturing method described in scope item (1).
菌のβ−ラクターゼのシグナルペプチドを有する発現ベ
クターを用いる特許請求の範囲 第（２）項に記載の方法。(3) The method according to claim (2), which uses an expression vector having a signal peptide of E. coli β-lactase as the gene encoding the signal peptide.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24909886A JPS63102695A (en) | 1986-10-20 | 1986-10-20 | Production of photoprotein aequorin using extracellular secretory vector of escherichia coli |
| GB8724178A GB2197652B (en) | 1986-10-20 | 1987-10-15 | Extracellular secretion of aequorin in escherichia coli |
| DE19873735380 DE3735380C2 (en) | 1986-10-20 | 1987-10-19 | Method for producing photoprotein equorin using an expression vector to be deposited on the outside of the bacterial body of Escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24909886A JPS63102695A (en) | 1986-10-20 | 1986-10-20 | Production of photoprotein aequorin using extracellular secretory vector of escherichia coli |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS63102695A true JPS63102695A (en) | 1988-05-07 |
Family
ID=17187938
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP24909886A Pending JPS63102695A (en) | 1986-10-20 | 1986-10-20 | Production of photoprotein aequorin using extracellular secretory vector of escherichia coli |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JPS63102695A (en) |
| DE (1) | DE3735380C2 (en) |
| GB (1) | GB2197652B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01132397A (en) * | 1987-11-18 | 1989-05-24 | Chisso Corp | Production of apoaequorin |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0376580A (en) * | 1989-08-17 | 1991-04-02 | Japan Tobacco Inc | Escherichia coli manifestation vector and production of antiviral protein using the same |
| CN107904254B (en) * | 2017-11-28 | 2021-11-23 | 大连大学 | Method for extracellular production of N-glycosylation recombinant protein by using escherichia coli |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61135586A (en) * | 1984-08-24 | 1986-06-23 | Chisso Corp | Plasmid containing luminescent protein aqualin, and biosynthesis of said luminescent protein |
| JPS61224989A (en) * | 1984-12-31 | 1986-10-06 | ユニヴア−シテイ・オブ・ジヨ−ジア・リサ−チ・フアウンデイシヨン・インコ−ポレイテツド | Recombined dna vector capable of developing apoecorine |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59176125A (en) * | 1983-03-26 | 1984-10-05 | Matsushita Electric Works Ltd | Manufacturing method of insulating trolley |
| JP2551746B2 (en) * | 1983-07-19 | 1996-11-06 | サントリー株式会社 | Improved plasmid vector and its use |
| DE3484950D1 (en) * | 1983-08-23 | 1991-09-26 | Mitsubishi Chem Ind | PLASMID VECTORS. |
| JPS6439990A (en) * | 1987-08-05 | 1989-02-10 | Chisso Corp | Fused gene by bonding aequorin gene to functional gene |
-
1986
- 1986-10-20 JP JP24909886A patent/JPS63102695A/en active Pending
-
1987
- 1987-10-15 GB GB8724178A patent/GB2197652B/en not_active Expired - Fee Related
- 1987-10-19 DE DE19873735380 patent/DE3735380C2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61135586A (en) * | 1984-08-24 | 1986-06-23 | Chisso Corp | Plasmid containing luminescent protein aqualin, and biosynthesis of said luminescent protein |
| JPS61224989A (en) * | 1984-12-31 | 1986-10-06 | ユニヴア−シテイ・オブ・ジヨ−ジア・リサ−チ・フアウンデイシヨン・インコ−ポレイテツド | Recombined dna vector capable of developing apoecorine |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01132397A (en) * | 1987-11-18 | 1989-05-24 | Chisso Corp | Production of apoaequorin |
| JP2592623B2 (en) * | 1987-11-18 | 1997-03-19 | チッソ株式会社 | Apoaequorin production method |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2197652A (en) | 1988-05-25 |
| DE3735380A1 (en) | 1988-04-21 |
| GB8724178D0 (en) | 1987-11-18 |
| DE3735380C2 (en) | 1994-03-10 |
| GB2197652B (en) | 1990-08-29 |
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