JPH0158950B2 - - Google Patents
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- Publication number
- JPH0158950B2 JPH0158950B2 JP58165065A JP16506583A JPH0158950B2 JP H0158950 B2 JPH0158950 B2 JP H0158950B2 JP 58165065 A JP58165065 A JP 58165065A JP 16506583 A JP16506583 A JP 16506583A JP H0158950 B2 JPH0158950 B2 JP H0158950B2
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- 108090000623 proteins and genes Proteins 0.000 claims description 33
- 239000013612 plasmid Substances 0.000 claims description 27
- 108020004414 DNA Proteins 0.000 claims description 23
- 241000193764 Brevibacillus brevis Species 0.000 claims description 10
- 102000018697 Membrane Proteins Human genes 0.000 claims description 8
- 108010052285 Membrane Proteins Proteins 0.000 claims description 8
- 230000001580 bacterial effect Effects 0.000 claims description 8
- 230000000694 effects Effects 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 5
- 238000003776 cleavage reaction Methods 0.000 claims description 4
- 230000007017 scission Effects 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 description 23
- 239000012634 fragment Substances 0.000 description 10
- 108091008146 restriction endonucleases Proteins 0.000 description 10
- 238000000034 method Methods 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 241001131785 Escherichia coli HB101 Species 0.000 description 7
- 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 5
- 229960000723 ampicillin Drugs 0.000 description 5
- 239000013611 chromosomal DNA Substances 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 102100032948 Aspartoacylase Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 101000797251 Homo sapiens Aspartoacylase Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102100040653 Tryptophan 2,3-dioxygenase Human genes 0.000 description 1
- 101710136122 Tryptophan 2,3-dioxygenase Proteins 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000126 substance Substances 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
Landscapes
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
Description
【発明の詳細な説明】
本発明はDNA、それを含むプラスミドおよび
該プラスミドを含む細菌細胞に関するものであ
る。
本発明者らは、バチルス・ブレビス（Bacillus
brevis）の表層蛋白質が菌体外に多量に生産され
ることに着目し、そのプロモーターを単離するこ
とを試み、遂にDNA鎖中にBamH1で切断され
る個所、その291bp下流にHinfで切断される個
所、その156bp下流にAluで切断される個所お
よびその268bp下流にHpaで切断される個所を
有し、バチルス・ブレビスの分子量15万の表層蛋
白質をコードする遺伝子のプロモーター活性を有
するDNAを分離することに成功したものである。
すなわち、ベクターとして大腸菌のプラスミド
pBR322を選択し、該プラスミドpBR322を制限
酵素Hindで切断し、5′末端のリン酸基をバク
テリアル・アルカリン・ホスフアターゼ
（Bacterial alkaline phosphatase）処理によつ
て除き線状のプラスミドpBR322を調製した。一
方、バチルス・ブレビス47菌の細胞から斉藤・三
浦の方法（Biochim.Biophys.Acta.72、619
（1963））によつて染色体DNAを調製してドナー
DNAに供した。この染色体DNAを制限酵素
Hindで部分加水分解して種々の長さの線状
DNAを調製した。次に、線状プラスミド
pBR322と線状DNAを混合し、T4DNAリガーゼ
で両者を連結した。連結したDNAを大腸菌
HB101にLederberg、E.M.and Cohen、S.N.の
方法（J.Bacteriol.、19、1072−1074（1974））に
より形質転換し、これをＬ−ブロス培地（トリプ
トン（Difco）１％、酵母エキス（Difco）0.5％、
NaCl0.5％、MgSO4・7H2O0.025％、PH7.2）に
アンピシリン50μｇ／mlを加えたものに培養して
アンピシリン耐性の形質転換株を得た。これらの
形質転換株からバチルス・ブレビス47の表層構造
蛋白質をコードする遺伝子を有するクローンを以
下の方法によつて選択した。
バチルス・ブレビス47の表層構造体は分子量13
万と分子量15万の主要蛋白質から構成されてい
る。これら蛋白質の混合物に対するウサギ抗体を
調製し、この抗血清を用いて上記形質転換株の中
から表層構造体を構成する蛋白質遺伝子を含むク
ローンをKemp、D.J.and Cowman、A.F.の方法
（Proc.Natl.Acad.Sci.USA、78、4520〜4524
（1981））により選択してNT100株を得た。この
クローンについて分子量13万と分子量15万の蛋白
質のそれぞれに対する抗体との反応性について検
討したところ分子量15万の表層構造蛋白質の抗体
とのみ反応する蛋白質を生産することが判つた。
第１図はNT100株に含まれるプラスミド
pNT100の制限酵素地図である。図中、太線部分
はバチルス・ブレビス47由来の染色体DNAを示
している。
次に、プラスミドpNT100を各種制限酵素
（Hind、Hpa、BamH）を用いて切断し、
再連結することによつて種々の長さのプラスミド
を調製した。これらプラスミドを用いて前記した
方法によつて大腸菌HB101を形質転換し、分子
量15万の表層蛋白質に対する抗体を用いて前記と
同様にして抗原抗体反応を行なつて分子量15万の
表層蛋白質を生産するクローンについて調べ、
pNT100中のプロモーター部位を決定した。その
結果、第１図におけるｂフラグメントに分子量15
万の表層蛋白質のプロモーター活性を示すDNA
配列が存在することが明らかとなつた。すなわ
ち、このDNAはDNA鎖中にBamHで切断され
る個所、その291bp下流にHinfで切断される個
所、その156bp下流にAluで切断される個所お
よびその268bp下流にHpaで切断される個所を
有している。
本発明のDNAは、細菌細胞、たとえば大腸菌
やバチルス属の微生物の細胞内で増殖することが
できるプラスミドに挿入すれば、活性の高いプロ
モーターとしての機能を発揮する。すなわち、プ
ラスミド内に挿入されたプロモーターDNAの下
流に有用な外来遺伝子を接続せしめ、得られた組
換えDNAを大腸菌またはバチルス属微生物の細
胞内に導入すれば、これら微生物は蛋白質等の有
用物質を多量に生産するように形質転換される。
ここで外来遺伝子の例としては真核生物由来のも
のおよび原核生物由来のもののいずれも使用する
ことが可能である。たとえばヒト遺伝子（たとえ
ばインターフエロン、インシユリンなど）、微生
物の酵素蛋白遺伝子（たとえばトリプトフアナー
ゼ、アスパラギン酸アンモニアリアーゼなど）な
どがある。
次に、本発明を実施例により説明する。
実施例
バチルス・ブレビス47菌（FERM Ｐ−7224）
の染色体DNAを制限酵素Hindで部分加水分解
し、種々の長さの線状DNAを調製した。一方、
プラスミドpBR322も制限酵素Hindで切断し、
5′末端のリン酸基をバクテリアル・アルカリ・ホ
スフアターゼ処理により除去して線状プラスミド
pBR322を調製した。
これら線状DNAを混合し、T4DNAリガーゼ
を作用させて両DNAを連結した。
連結したDNAを大腸菌HB101にLederberg、
E.M.and Cohen、S.N.の方法（J.Bacteriol.、
119、1072〜1074（1974））によつて形質転換し、
さらにＬ−broth培地にアンピシリン50μｇ／ml
を加えた培地に塗布して培養し、アンピシリン耐
性の形質転換株を得た。
次に、バチルス・ブレビス47菌の表層構造体を
構成する分子量13万と15万の蛋白質の混合物に対
するウサギ抗体を調製し、この抗血清を使用して
上記形質転換株中より表層構造体構成蛋白質遺伝
子を含むクローンをKemp、D.J.and Cowman、
A.F.の方法（Proc.Natl.Acad.Sci.USA、78、
4520〜4524（1981））により選択してNT100株を
得た。このNT100株について分子量13万と15万
のそれぞれの蛋白質に対する抗体との反応性を検
討したところ、この菌株は分子量15万の表層構造
蛋白質の抗体とのみ反応する蛋白質を生産してい
ることが明らかとなつた。
このNT100株を用いてBirnboim、H.C.、
Doly、J.（Nucleic Acids Res.、７、1513−1523
（1979））の方法によりプラスミドの検出および分
子量の測定を行なつた。その結果、分子量は6.1
×106ダルトンであり、このプラスミドを
pNT100と命名した。pNT100の制限酵素切断地
図は第１図に示した通りである。
プラスミドpNT100を各種制限酵素（Hind、
Hpa、BamH）を用いて切断し、再連結す
ることにより種々の長さのプラスミド７種を調製
し、これらのプラスミドを上記した方法により大
腸菌HB101に形質転換し、７種類のクローンを
得、分子量15万の表層構造蛋白質に対する抗体を
用いて上記方法と同様にして抗原抗体反応を行な
い、分子量15万の表層構造蛋白質のプロモーター
部位を決定した。以下にその詳細を示す。
(1) Hindを用いた実験
プラスミドpNT100をHindで切断し、再
連結を行なうと、3KbのHindDNAフラグメ
ントだけで分子量15万の蛋白質の抗体と反応す
る蛋白質を大腸菌は生産した（第２図Ｂ）。本
菌Escherichia coli HB101／pNT100は微工
研にFERM Ｐ−7231として寄託されている。
(2) Hpaを用いた実験
プラスミドpNT100をHpaで切断し、再連
結し、分子量15万の蛋白質の抗体と反応する蛋
白質を生産する大腸菌の中に含まれるプラスミ
ドを分析した結果、第２図Ｃ，Ｄ，Ｅの３種類
の株が得られた。すなわち、第１図に示したａ
フラグメント（Hpa−BamH、400bp）、
ｂフラグメント（BamH−Hpa、715bp）
およびｃフラグメント（Hpa−Hpa、
213bp）の連結の仕方が種々異なつていた。ク
ローンＤではａ、ｂ、ｃのフラグメントの連結
の仕方はpNT100と全く逆方向であり、かつ分
子量15万の蛋白質の抗体と反応する蛋白質を生
産していた。また、クローンＥではｃフラグメ
ントが欠けていたが、クローンＤと同様に分子
量15万の蛋白質の抗体と反応する蛋白質を生産
していた。したがつて、分子量15万の蛋白質の
プロモーターはａ、ｂフラグメント中に存在す
ることが明らかである。
(3) BamH、Hpa両制限酵素を用いた実験
プラスミドpNT100をBamHとHpaで切
断し、再連続して分子量15万の蛋白質の抗体と
反応する蛋白質を生産する大腸菌としてクロー
ンＦ（本菌Ｅ．coli HB101／pNT132は微工
研にFERM Ｐ−7230として寄託されている。）
が得られた。第２図に示したように、クローン
Ｆはａフラグメントが欠けているが、分子量15
万の蛋白質の抗体と反応する蛋白質を生産して
いる。また、ｂフラグメントのみを有するプラ
スミドpNT142を導入したクローンＧ（本菌Ｅ．
coli HB101／pNT142）は微工研にFERM
Ｐ−7229として寄託されている。
以上のように、クローンＡ〜Ｇはいずれも分子
量15万の蛋白質の抗体との反応性が陽性である蛋
白質を生成する。これらの結果に基いて総合的に
判断すると、第１図におけるｂフラグメントに分
子量15万の蛋白質のプロモーター活性を示す
DNA配列が存在する。 DETAILED DESCRIPTION OF THE INVENTION The present invention relates to DNA, plasmids containing the same, and bacterial cells containing the plasmids. The present inventors have discovered that Bacillus brevis (Bacillus brevis)
Noting that the surface protein of brevis (brevis) is produced in large quantities outside the bacterial cell, we attempted to isolate its promoter, and finally found a site in the DNA strand that was cleaved with Hinf, 291 bp downstream of the site that was cleaved with BamH1. DNA that has a site that is cleaved with Alu 156 bp downstream and a site that is cleaved with Hpa 268 bp downstream, and has promoter activity of a gene encoding a surface protein of Bacillus brevis with a molecular weight of 150,000 was isolated. It was successful in doing so. i.e., an E. coli plasmid as a vector.
pBR322 was selected, the plasmid pBR322 was cut with the restriction enzyme Hind, and the 5'-end phosphate group was removed by bacterial alkaline phosphatase treatment to prepare a linear plasmid pBR322. On the other hand, the method of Saito and Miura (Biochim. Biophys. Acta. 72 , 619
(1963)) to prepare donor chromosomal DNA.
Subjected to DNA. This chromosomal DNA is digested with restriction enzymes.
Lines of various lengths are partially hydrolyzed with Hind.
DNA was prepared. Next, the linear plasmid
pBR322 and linear DNA were mixed and ligated together using T 4 DNA ligase. Insert the linked DNA into E. coli
HB101 was transformed by the method of Lederberg, EMand Cohen, SN (J. Bacteriol., 19 , 1072-1074 (1974)), and this was transformed into L-broth medium (1% tryptone (Difco), 0.5% yeast extract (Difco)). %,
Ampicillin-resistant transformants were obtained by culturing in 0.5% NaCl, 0.025% MgSO 4 .7H 2 O, PH 7.2) to which 50 μg/ml of ampicillin was added. From these transformed strains, clones containing the gene encoding the surface structure protein of Bacillus brevis 47 were selected by the following method. The surface structure of Bacillus brevis 47 has a molecular weight of 13
It is composed of major proteins with a molecular weight of 150,000 and 150,000. A rabbit antibody against a mixture of these proteins was prepared, and using this antiserum, clones containing protein genes constituting the surface structure were isolated from among the above-mentioned transformed strains using the method of Kemp, DJ and Cowman, AF (Proc. Natl. Acad. .Sci.USA, 78 , 4520–4524
(1981)) to obtain the NT100 strain. When this clone was examined for its reactivity with antibodies to proteins with a molecular weight of 130,000 and 150,000, it was found that it produced a protein that reacted only with antibodies to surface structure proteins with a molecular weight of 150,000. Figure 1 shows the plasmid contained in the NT100 strain.
This is a restriction enzyme map of pNT100. In the figure, the bold line portion indicates chromosomal DNA derived from Bacillus brevis47. Next, plasmid pNT100 was cut using various restriction enzymes (Hind, Hpa, BamH).
Plasmids of various lengths were prepared by religation. E. coli HB101 is transformed using these plasmids by the method described above, and an antigen-antibody reaction is performed in the same manner as above using an antibody against the surface protein with a molecular weight of 150,000 to produce a surface protein with a molecular weight of 150,000. Find out about clones
The promoter site in pNT100 was determined. As a result, the b fragment in Figure 1 has a molecular weight of 15
DNA showing promoter activity of surface protein of 10,000
It became clear that a sequence existed. That is, this DNA has a site in the DNA strand that is cut with BamH, a site that is cut with Hinf 291 bp downstream of that, a site that is cut with Alu 156 bp downstream of that, and a site that is cut with Hpa 268 bp downstream of that. are doing. When the DNA of the present invention is inserted into a plasmid that can proliferate in bacterial cells, such as Escherichia coli or microorganisms belonging to the genus Bacillus, it will function as a highly active promoter. In other words, if a useful foreign gene is connected downstream of the promoter DNA inserted into a plasmid and the resulting recombinant DNA is introduced into the cells of Escherichia coli or Bacillus microorganisms, these microorganisms will be able to produce useful substances such as proteins. Transformed to produce large quantities.
Here, as examples of foreign genes, both those derived from eukaryotes and those derived from prokaryotes can be used. Examples include human genes (eg, interferon, insulin, etc.) and microbial enzyme protein genes (eg, tryptophanase, aspartate ammonia lyase, etc.). Next, the present invention will be explained by examples. Example Bacillus brevis 47 bacteria (FERM P-7224)
Chromosomal DNA was partially hydrolyzed with the restriction enzyme Hind to prepare linear DNA of various lengths. on the other hand,
Plasmid pBR322 was also cut with the restriction enzyme Hind,
The 5′-terminal phosphate group was removed by bacterial alkaline phosphatase treatment to create a linear plasmid.
pBR322 was prepared. These linear DNAs were mixed and T 4 DNA ligase was applied to connect both DNAs. The ligated DNA was transferred to E. coli HB101 by Lederberg.
EMand Cohen, SN's method (J. Bacteriol.,
119, 1072-1074 (1974)),
Furthermore, ampicillin 50 μg/ml was added to L-broth medium.
The cells were spread on a medium supplemented with ampicillin and cultured to obtain ampicillin-resistant transformed strains. Next, a rabbit antibody against a mixture of proteins with molecular weights of 130,000 and 150,000, which constitute the surface structure of 47 Bacillus brevis bacteria, was prepared, and this antiserum was used to isolate the proteins that constitute the surface structure from the above-mentioned transformed strain. A clone containing the gene was created by Kemp, DJand Cowman,
Methods of AF (Proc.Natl.Acad.Sci.USA, 78 ,
4520-4524 (1981)) to obtain the NT100 strain. When we examined the reactivity of this NT100 strain with antibodies to proteins with a molecular weight of 130,000 and 150,000, it was clear that this strain produced a protein that only reacted with antibodies to surface structure proteins with a molecular weight of 150,000. It became. Using this NT100 strain, Birnboim, HC,
Doly, J. (Nucleic Acids Res., 7 , 1513-1523
(1979)) was used to detect the plasmid and measure its molecular weight. As a result, the molecular weight is 6.1
x10 6 daltons and this plasmid
It was named pNT100. The restriction enzyme cleavage map of pNT100 is shown in FIG. Plasmid pNT100 was digested with various restriction enzymes (Hind,
Seven types of plasmids of various lengths were prepared by cutting and religating using Hpa, BamH), and these plasmids were transformed into E. coli HB101 by the method described above, seven types of clones were obtained, and the molecular weight An antigen-antibody reaction was performed in the same manner as above using an antibody against a surface structure protein with a molecular weight of 150,000, and the promoter site of a surface structure protein with a molecular weight of 150,000 was determined. The details are shown below. (1) Experiment using Hind When plasmid pNT100 was cut with Hind and religated, E. coli produced a protein with a molecular weight of 150,000 that reacted with the antibody using just the 3 Kb Hind DNA fragment (Figure 2B). . The present bacterium, Escherichia coli HB101/pNT100, has been deposited with the Microtech Institute as FERM P-7231. (2) Experiment using Hpa Plasmid pNT100 was cut with Hpa and religated, and the results of analyzing the plasmid contained in E. coli, which produces a protein that reacts with an antibody with a molecular weight of 150,000, were shown in Figure 2C. , D, and E were obtained. That is, a shown in Figure 1
Fragment (Hpa−BamH, 400bp),
b fragment (BamH-Hpa, 715bp)
and c fragment (Hpa−Hpa,
213bp) were linked in various ways. In clone D, the way in which fragments a, b, and c were linked was completely opposite to that of pNT100, and a protein with a molecular weight of 150,000 that reacted with an antibody was produced. Furthermore, clone E lacked the c fragment, but like clone D, it produced a protein with a molecular weight of 150,000 that reacts with antibodies. Therefore, it is clear that the promoter of a protein with a molecular weight of 150,000 exists in the a and b fragments. (3) Experiment using both BamH and Hpa restriction enzymes Plasmid pNT100 was cut with BamH and Hpa, and the E. coli clone F (this bacterium E. coli HB101/pNT132 has been deposited with the Microtech Institute as FERM P-7230.)
was gotten. As shown in Figure 2, clone F lacks the a fragment, but has a molecular weight of 15
It produces proteins that react with antibodies from 10,000 proteins. In addition, clone G (this bacterium E.
coli HB101/pNT142) was FERMed to the Microtech Institute.
It has been deposited as P-7229. As described above, clones A to G all produce proteins with a molecular weight of 150,000 that are positively reactive with antibodies. Judging comprehensively based on these results, the b fragment in Figure 1 shows the promoter activity of a protein with a molecular weight of 150,000.
DNA sequences exist.
第１図はプラスミドpNT100の制限酵素地図、
第２図は大腸菌に導入された各種のプラスミドを
示す。
Figure 1 shows the restriction enzyme map of plasmid pNT100.
Figure 2 shows various plasmids introduced into E. coli.
Claims (1)
の291bp下流にHinfで切断される個所、その
156bp下流にAluで切断される個所およびその
268bp下流にHpaで切断される個所を有し、バ
チルス・ブレビスの分子量15万の表層蛋白質をコ
ードする遺伝子のプロモーター活性を有する
DNA。 ２ DNA鎖中にBamH1で切断される個所、そ
の291bp下流にHinfで切断される個所、その
156bp下流にAluで切断される個所およびその
268bp下流にHpaで切断される個所を有し、バ
チルス・ブレビスの分子量15万の表層蛋白質をコ
ードする遺伝子のプロモーター活性を有する
DNAを含む細菌細胞内で増殖しうるプラスミド。 ３ DNA鎖中にBamH1で切断される個所、そ
の291bp下流にHinfで切断される個所、その
156bp下流にAluで切断される個所およびその
268bp下流にHpaで切断される個所を有し、バ
チルス・ブレビスの分子量15万の表層蛋白質をコ
ードする遺伝子のプロモーター活性を有する
DNAを含む細菌細胞内で増殖しうるプラスミド
を含む細菌細胞。[Claims] 1. A site in the DNA strand that is cleaved with BamH1, a site 291 bp downstream of that that is cleaved with Hinf, and
156bp downstream of the Alu cleavage site and its
It has a site that is cleaved by Hpa 268bp downstream and has promoter activity of the gene encoding the surface protein of Bacillus brevis with a molecular weight of 150,000.
DNA. 2. The site in the DNA strand that is cut by BamH1, the site 291bp downstream that is cut by Hinf, and the
156bp downstream of the Alu cleavage site and its
It has a site that is cleaved by Hpa 268bp downstream and has promoter activity of the gene encoding the surface protein of Bacillus brevis with a molecular weight of 150,000.
A plasmid that can grow in bacterial cells that contains DNA. 3 The site in the DNA strand that is cut by BamH1, the site 291 bp downstream that is cut by Hinf, and the
156bp downstream of the Alu cleavage site and its
It has a site that is cleaved by Hpa 268bp downstream and has promoter activity of the gene encoding the surface protein of Bacillus brevis with a molecular weight of 150,000.
A bacterial cell that contains a plasmid that can grow within a bacterial cell that contains DNA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58165065A JPS6058073A (en) | 1983-09-09 | 1983-09-09 | Dna |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP58165065A JPS6058073A (en) | 1983-09-09 | 1983-09-09 | Dna |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS6058073A JPS6058073A (en) | 1985-04-04 |
| JPH0158950B2 true JPH0158950B2 (en) | 1989-12-14 |
Family
ID=15805192
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP58165065A Granted JPS6058073A (en) | 1983-09-09 | 1983-09-09 | Dna |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6058073A (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07108224B2 (en) * | 1985-11-07 | 1995-11-22 | 重三 鵜高 | Gene expression method using Bacillus brevis |
| EP0326046A3 (en) * | 1988-01-25 | 1990-06-13 | Takeda Chemical Industries, Ltd. | Production of human epidermal growth factor |
-
1983
- 1983-09-09 JP JP58165065A patent/JPS6058073A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS6058073A (en) | 1985-04-04 |
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