JPH0272882A - Expression vector - Google Patents
Expression vectorInfo
- Publication number
- JPH0272882A JPH0272882A JP22594788A JP22594788A JPH0272882A JP H0272882 A JPH0272882 A JP H0272882A JP 22594788 A JP22594788 A JP 22594788A JP 22594788 A JP22594788 A JP 22594788A JP H0272882 A JPH0272882 A JP H0272882A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- promoter region
- expression vector
- plasmid
- candida maltosa
- 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.)
- Pending
Links
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Abstract
Description
【発明の詳細な説明】
本発明は、無胞子酵母キャンディダ、マル1−−サ(C
andida maltosa )の細胞内に於いて、
外来遺伝子を発現させるための発現ベクターに関する。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of the nonspore-free yeast Candida, Mal 1-sa (C
andida maltosa),
This invention relates to an expression vector for expressing a foreign gene.
従来組換えDNAの宿主としては、主として大腸菌に1
2株(EK系)、枯草菌(BS系)及びサツカロマイセ
ス・セレビシェ(Saccharomycescere
visiae) (S C系)が汎用されてきたが、
例えば、ある遺伝子産物がEK系では不安定で分解を受
けやすかったり、宿主に毒性を示したりするが、SC系
では安定に生産されるといった知見も蓄積されてきつつ
ある。Conventionally, Escherichia coli is the main host for recombinant DNA.
2 strains (EK system), Bacillus subtilis (BS system) and Saccharomyces cerevisiae (Saccharomyces scere).
visiae) (SC series) has been widely used,
For example, knowledge is accumulating that certain gene products are unstable and easily degraded or toxic to the host in the EK system, but are stably produced in the SC system.
従って、有用物質の大量生産を目的とする場合、宿主細
胞の選定も重要なポイントとなり、この、官味で宿主の
選択域を広げておく事は重要な意義を有している。現在
では上記の宿主以外に、(Acet。Therefore, when the purpose is mass production of useful substances, the selection of host cells is also an important point, and expanding the range of host selection based on taste has important significance. Currently, in addition to the above-mentioned hosts, (Acet.
culis (CH系) 、 Pseudomona
s utida (P P系)ii(ZR系)等も用
いられる様になってきている。Culis (CH series), Pseudomona
sutida (PP series) ii (ZR series) and the like are also coming into use.
本発明者らは、さらに宿主の選択域を広げるべく、n−
アルカンの資化能、尿酸の分解能等の特殊な有機化合物
の代謝能を有するキャンデイグ3包訓牡鎖)属の酵母に
注目し、n−アルカンの資化能に優れた特性を示すギャ
ンディダ・マルトーサ(Candida maltos
a)を有用物質の生産や産業廃棄物の処理等に応用する
事を目的として、キャンディダ・マルトーサ(Cand
ida maltosa)の宿主ベクター系の開発を行
ってきた(例えばTakagiら、J。In order to further expand the range of host selection, the present inventors investigated n-
We focused on the yeast of the genus Candida, which has the ability to metabolize special organic compounds such as the ability to assimilate alkanes and decompose uric acid. (Candida maltos
With the aim of applying a) to the production of useful substances and the treatment of industrial waste, Candida maltosa (Candida maltosa)
ida maltosa) have been developed (e.g., Takagi et al., J.
Bacteriol、167551 (1986) ;
Takagiら、Curr、Genet、ll 451
(1987) i特開昭62−74287及び特開昭
6274288参照)。Bacteriol, 167551 (1986);
Takagi et al., Curr, Genet, ll 451
(1987) i JP-A-62-74287 and JP-A-6274288).
一方、ある宿主細胞に於いて外来遺伝子を効率良く発現
させるためには、転写開始に必要な遺伝子シグナルであ
るプロモーターの存在や、そのプロモーター活性の強弱
が重要な役割を果している事が知られているが、キャン
ディダ・マルトーサ(Candida maltosa
)の遺伝子のプロモーターについてはほとんど知見がな
いのが現状である(例えば、Takagiら、Curr
、Genet、 11451 (1987) )。On the other hand, it is known that the presence of a promoter, which is a gene signal necessary for transcription initiation, and the strength of the promoter activity play important roles in order to efficiently express a foreign gene in a host cell. However, Candida maltosa
) Currently, there is little knowledge about the promoter of the gene (for example, Takagi et al., Curr
, Genet, 11451 (1987)).
本発明者らは、この様な現状にかんがみ、キャンディダ
・マルトーサ(Candida maltosa)の細
胞内で機能するプロモーターを取得するためには先ず、
プロモーター検索用プラスミドヘクターを開発する必要
があると考え、ブロモ−クー検索用プラスミドヘクタ−
pPLc1及びpPLC2(特願昭 62−19896
2)を開発し、キャンディダ・マルトーサの染色体DN
Aより、炭素源がグルコース、グリセロール及びテトラ
デカンの時に強い活性を示すプロモーターを含むDNA
断片及び炭素源がグルコースの場合には活性が抑制され
るが、グリセロール及びテトラデカンの時に強い活性を
示すプロモータを含むDNA断片を取得した。In view of the current situation, the present inventors first determined that in order to obtain a promoter that functions in Candida maltosa cells,
Considering the need to develop a plasmid hector for promoter search, we developed a plasmid hector for promoter search.
pPLc1 and pPLC2 (patent application 1989-19896)
2) was developed, and the chromosomal DNA of Candida maltosa was
From A, DNA containing a promoter that exhibits strong activity when the carbon source is glucose, glycerol, and tetradecane.
We obtained a DNA fragment containing a promoter whose activity was suppressed when the fragment and carbon source were glucose, but showed strong activity when glycerol and tetradecane were used.
この様にして得られたDNA断片を利用して発現ベクタ
ーを開発すべく鋭意、検討、工夫を重ねた結果、本発明
の発現ベクターが、外来遺伝子をキャンディダ・マルト
ーサの細胞内に於いて発現させるための発現ベクターと
して優れたものである事を確認し、本発明を完成するに
至った。As a result of repeated efforts, studies, and ingenuity to develop an expression vector using the DNA fragments obtained in this way, the expression vector of the present invention is capable of expressing a foreign gene in Candida maltosa cells. It was confirmed that the vector was excellent as an expression vector for the expression, and the present invention was completed.
本発明は、従来の宿主細胞にはみられない有用な特性で
あるn−アルカン資化能を有するキャンディダ・マルト
ーサを宿主細胞とする新規な宿主・ベクター系に於いて
、外来遺伝子産物を大量に取得するための発現ベクター
を提供するものである。The present invention utilizes a novel host-vector system using Candida maltosa as a host cell, which has the ability to assimilate n-alkanes, which is a useful property not found in conventional host cells. The present invention provides an expression vector for obtaining the desired expression vector.
本発明の発現ベクターは、
(1)キャンディダ・マルトーサの細胞内において機能
する複製開始点
(2)キャンディダ・マルトーサの染色体DNA由来の
プロモーター領域
(3)上記プロモーター領域下流に設けられた制限酵素
切断部位
(4)サツカロマイセス・セレビシェのLEU2遺伝子
由来のターミネータ−領域
を含存する事によって特徴づけられるが、さらにプラス
ミドの調整等を容易にするために大腸菌中でも複製、選
択ができる様になっている大腸菌のシャトルベクターと
しての特徴も有している。The expression vector of the present invention includes (1) a replication origin that functions in cells of Candida maltosa, (2) a promoter region derived from the chromosomal DNA of Candida maltosa, and (3) a restriction enzyme provided downstream of the promoter region. Cleavage site (4) Escherichia coli is characterized by containing the terminator region derived from the LEU2 gene of Satucharomyces cerevisiae, but is also capable of replication and selection in Escherichia coli to facilitate plasmid adjustment, etc. It also has the characteristics of a shuttle vector.
キャンディダ・マルトーサの細胞内において機能する複
製開始点とは、該細胞中に於いてプラスミドベクターが
複製するために必要な機能を司るDN A nM域を表
わすものであるが、キャンディダ・マルトーサからは今
だプラスミドが分離されていないため、該細胞の染色体
DNAよりその様な機能を存するDNA領域を調製して
くる必要がある。The replication origin that functions in Candida maltosa cells represents a DNA nM region that controls the functions necessary for the plasmid vector to replicate in the cells. Since a plasmid has not yet been isolated, it is necessary to prepare a DNA region having such a function from the chromosomal DNA of the cell.
キャンディダ・マルトーサの染色体DNAより、その様
なり N A ?+Jl域を調整する方法としては、既
にその一例が文献に開示されており、それに従って行う
事ができるが、既にその様なりNA領領域組み込まれた
組換えプラスミド例えばpTl?^1(例えば、Tak
agiら、J、uacteriol、16755H19
86)及び特開昭62−74288参照) 、pptc
t及びpPLC2(特願昭62−198962参照)等
を利用するのが簡便である。That's what the chromosomal DNA of Candida maltosa looks like.NA? An example of a method for adjusting the +Jl region has already been disclosed in the literature, and it can be carried out according to that method. ^1 (for example, Tak
agi et al., J. uacteriol, 16755H19
86) and JP-A-62-74288), pptc
It is convenient to use t and pPLC2 (see Japanese Patent Application No. 62-198962).
キャンディダ・マルトーサの染色体DNA由来のプロモ
ーター領域とは、当38 SR域上下流外来遺伝子を挿
入した時に、当該遺伝子の転写を可能にするための遺伝
子シグナルを荷う領域を表わすものであり、その様な領
域は、キャンディダ・マルトーサの染色体DNAを制限
酵素による消化等の適当な手段で切断し、プロモーター
検索用プラスミドベクターpPLc1又はppt、c2
に挿入して、ロイシン要求性のキャンディダ・マルトー
サ(例えば3288株)細胞に導入し、ロイシン要求性
の解除された形質転換体を選択して、当該形質転換体よ
りプラスミドを回収する事によって取得する事ができる
(特願昭62−198962参照)。The promoter region derived from the chromosomal DNA of Candida maltosa represents the region that carries the gene signal that enables transcription of the gene when the foreign gene upstream and downstream of the 38 SR region is inserted. Such regions can be obtained by cleaving the chromosomal DNA of Candida maltosa by appropriate means such as digestion with restriction enzymes, and creating plasmid vectors pPLc1, ppt, and c2 for promoter search.
The plasmid is obtained by inserting it into leucine-requiring Candida maltosa (e.g., strain 3288) cells, selecting transformants in which the leucine-requiring requirement has been removed, and collecting the plasmid from the transformants. (See Japanese Patent Application No. 62-198962).
上記プロモーター領域下流に設けられた制限酵素切断部
位とは、外来遺伝子を組み込むための切断部位であり、
制限酵素はどの様なものでも良いが、通常汎用されてい
るものの内で当該発現ベクターの他の部分には切断部位
のないもの、例えば、Smal等が好便である。The restriction enzyme cleavage site provided downstream of the promoter region is a cleavage site for incorporating a foreign gene,
Any restriction enzyme may be used, but among the commonly used restriction enzymes, those that do not have a cleavage site in other parts of the expression vector, such as Smal, are convenient.
制限酵素の切断部位は、例えば、市販の適当な合成リン
カ−を挿入する事等によって容易に創製する事ができる
。A restriction enzyme cleavage site can be easily created, for example, by inserting a commercially available appropriate synthetic linker.
サツカロマイセス・セレビシェのL E U 2 A転
子由来のターミネータ−領域とは、遺伝子の転写を停止
させるための遺伝子シグナルを荷なう領域を表すもので
あり、それにより外来遺伝子の発現を効率的に行わせる
ことができる。その様な領域はサツカロマイセス・セレ
ビシェの染色体DNAより調整する事も可能であるが、
すでにその様な領域を含むプラスミド、例えばYEp
13 (Broachら、Gene 8121 (19
79)参照)等から調整するのが軽便である。The terminator region derived from the L EU 2 A trochanter of Satucharomyces cerevisiae represents a region that carries a gene signal to stop gene transcription, thereby efficiently expressing foreign genes. You can make it happen. Although such a region can be adjusted from the chromosomal DNA of Satucharomyces cerevisiae,
A plasmid that already contains such a region, e.g. YEp
13 (Broach et al., Gene 8121 (19
It is easy to adjust from 79) etc.
プラスミドの構築、調製等を行うには、大腸菌を宿主と
するのがより簡便であるため、大腸菌中でも復製、選択
が可能なシャトルベクターとして構築するのが望ましい
。Since it is easier to construct and prepare a plasmid using E. coli as a host, it is preferable to construct it as a shuttle vector that can be reproduced and selected in E. coli.
大IIa菌中で複製、選択が可能なベクターとしては、
種りの公知、のベクター、例えばpUclB(Mess
ing、Methods in EnzyIIIolo
gy 1012O−78(1983))等を利用する事
ができる。Vectors that can be replicated and selected in E. IIa include:
Seed vectors such as pUclB (Mess
ing, Methods in EnzyIIIolo
gy 1012O-78 (1983)) etc. can be used.
本発明の発現ベクターは、次の様にして構築する事がで
きる。キャンディダ・マルトーサの染色体DNAを5a
u3AIで切断し、約1〜2KbのDNA断片を得、こ
の断片をpPLc:1のBal1)II切断部位に組み
込み、キャンディダ・マルトーサ3288株(特願昭6
2−198962参照)を形質転換する。形質転換体を
グルコース、グリセロール又はテトラデカンを炭素源と
する最少培地上で30°C110日間生育させ、炭素源
がグルコース、グリセロール及びテトラデカンのいずれ
の場合にも生育する形質転換体からプラスミドDNAを
回収し、第1図に示される様な挿入断片を含むpcP−
AIを得る。又炭素源がグルコースの場合には生育しな
いがグリセロール及びテトラデカンの場合には生育する
形質転換体よりプラスミドDNAを回収し第1図に示さ
れる様な挿入断片を含むpCP−81を得る。pCPA
[を1lae I[、又pCP−81を1(as II
及び[1gl [で切断し、プロモーター領域を含む各
々約0.9Kb及び2.OKbのDNA断片を回収し、
pU−C191ES(pUc19をEcoRl及びSm
a lで切断後再連結してEcoR1及びSma■切断
部位を消失させたプラスミド)の[lam旧切断部位に
挿入して第3図に示される様なpUcP−AI (3,
6Kb)及びpUcP−Bl(4,7Kb)を得る。The expression vector of the present invention can be constructed as follows. 5a chromosomal DNA of Candida maltosa
A DNA fragment of about 1 to 2 Kb was obtained by cutting with u3AI, and this fragment was inserted into the Bal1) II cleavage site of pPLc:1 to create Candida maltosa strain 3288 (patent application No. 6).
2-198962)). The transformants were grown for 110 days at 30°C on a minimal medium containing glucose, glycerol, or tetradecane as the carbon source, and plasmid DNA was recovered from the transformants that grew when the carbon source was glucose, glycerol, or tetradecane. , pcP- containing the insert as shown in FIG.
Get AI. Plasmid DNA is recovered from transformants that do not grow when the carbon source is glucose but grow when glycerol and tetradecane are used, and pCP-81 containing the inserted fragment as shown in FIG. 1 is obtained. pCPA
[as II], and pCP-81 as II
and [1gl], each containing approximately 0.9 Kb and 2.9 Kb including the promoter region. Collect OKb DNA fragments,
pU-C191ES (pUc19 with EcoRl and Sm
pUcP-AI (3,
6 Kb) and pUcP-Bl (4,7 Kb).
次に、pUcP−^l及びpUcP−BlをBstEn
で切断し、ρPLCI由来のLEU2遺伝子領域を欠失
させ、Sma Iリンカ−を連結した後、Baa旧及び
Sea 1で切断してプロモーター領域を含む約0.6
にb及び約1.7 KbのDNA断片を得る。このDN
A断片を、P+JCT1 (YEp13をXho I及
び5ailで切断して得られるすνカリマイセス・セレ
ビシェ(Saccharom匹as 5erevist
ae)のに旦旦2遺伝子を含むDNA断片をpUclB
のXho I切断部位に挿入してpt1118を得、p
UL18をFok Iで切断し、Sma Iリンカ−を
連結した後Sal 1で切断して旦旦旦2遺伝子のター
ミネータ−領域を含む約0.3KbのDNA断片を得る
。次にこのDNA断片をρUC19をSma l及びS
at [で切断した部位に挿入し、さらにマルチプル
・クローニング・サイトのEco RI切ILJ位置上
流にBa渭HI切断部位を導入して得られたプラスミド
)をBaIIIII及びS+ma Iで切断した部位に
挿入し第3図に示される様なプラスミドpSC−AI
(3,6Xb)及びpSC−81(4,7Kb)を得る
。Next, pUcP-^l and pUcP-Bl were added to BstEn
After cutting with Baa old and Sea 1 to delete the LEU2 gene region derived from ρPLCI and ligating a Sma I linker, it was cut with Baa old and Sea 1 to create a gene containing the promoter region of about 0.6
A DNA fragment of approximately 1.7 Kb was obtained. This DN
The A fragment was obtained by cleaving P+JCT1 (YEp13 with Xho I and 5ail).
ae), the DNA fragment containing the two genes was transformed into pUclB
into the Xho I cleavage site of p
UL18 is cut with Fok I, ligated with a Sma I linker, and then cut with Sal 1 to obtain a DNA fragment of approximately 0.3 Kb containing the terminator regions of the two genes. Next, this DNA fragment was divided into ρUC19, Sma I and S
[A plasmid obtained by inserting the plasmid into the site cut with BaIII and S+ma I and further introducing the Ba Wei HI cut site upstream of the Eco RI cut ILJ position of the multiple cloning site] was inserted into the site cut with BaIII and S+ma I. Plasmid pSC-AI as shown in Figure 3
(3,6Xb) and pSC-81 (4,7Kb).
さらに、pSC−AI及びpSC−BlをBaa II
I及びSal+で切断して得られるプロモーター及びタ
ーミネータ−領域を含む約0.9Kb及び約2. OK
bのDNA断片を、pBT810B (キャンデイダ
・マルトーサ由来のT RA fil域(Takagi
ら、J、Bacteriol、167551(1986
)、特開昭62−74288及び特願昭62−198’
162参照]をpBR322のEco Rr切断部位に
挿入した後、T RA ?iJ¥域内のXho I切
断部位にc−H[S(ヒスチジン要求性のキャンディダ
・マルトーサ(Changら、J、Gen’、^pp1
.Microbio1.30189 (1984)参照
)の栄養要求性を相補するキャンディダ・マルトーサI
AM12247株由来のDNA断片〕を挿入して得られ
たプラスミド)のBglff切 断部位に挿入して第4
図に示される様なプラスミドpCEV−AI (11゜
4Kb)及びpCEV−Bl (12,5Kb)を得る
。Furthermore, pSC-AI and pSC-Bl were transformed into Baa II
Approximately 0.9 Kb and approximately 2.0 Kb containing promoter and terminator regions obtained by cutting with I and Sal+. OK
The DNA fragment of b was inserted into pBT810B (TRA fil region derived from Candida maltosa (Takagi).
et al., J. Bacteriol, 167551 (1986
), Japanese Patent Application Publication No. 62-74288 and Japanese Patent Application No. 62-198'
162] into the Eco Rr cleavage site of pBR322, then T RA ? The Xho I cleavage site within the iJ\ region contains c-H[S (histidine-requiring Candida maltosa (Chang et al., J. Gen', ^pp1
.. Microbio1.30189 (1984)) which complements the auxotrophy of Candida maltosa I
A DNA fragment derived from strain AM12247] was inserted into the Bglff cleavage site of the plasmid obtained by inserting the DNA fragment derived from strain AM12247.
Plasmids pCEV-AI (11°4 Kb) and pCEV-Bl (12,5 Kb) as shown in the figure are obtained.
キャンディダ・マルトーサの宿主細胞としては、キャン
ディダ・マルトーサIAM 12247株より誘導され
たヒスチジン要求性変異株CHI株等を用いる事ができ
る。キャンディダ・マルトーサCHI株は微工研菌寄第
10173号(FERMP−10173)の番号のもと
に微生物工業技術研究所に寄託されている。また本発明
のプラスミドベクターはサツカロマイセス・セレビシェ
及びニジエリシア・コリ(Escherichia c
olk)を宿主として利用する事も可能である(Tak
agi ら、J、Bacteriol、 16755H
1986);特開昭62−14281及び特開昭62−
14288> 。As a host cell for Candida maltosa, a histidine auxotrophic mutant strain CHI derived from Candida maltosa IAM 12247 strain can be used. Candida maltosa strain CHI has been deposited at the National Institute of Microbial Technology under the number FERMP-10173. Furthermore, the plasmid vector of the present invention can be used for Saccharomyces cerevisiae and Escherichia coli (Escherichia coli).
olk) can be used as a host (Tak
agi et al., J. Bacteriol, 16755H
1986); JP-A-62-14281 and JP-A-62-
14288>.
宿主細胞の形質転換、宿主細胞からのDNAの調製等は
、公知の常法に従って行う事ができる(Ilinnen
ら、Proc、Natl、^cad、sci、UsA
751929 (1978)及びItoら、J、Bac
teriol、153165(1983)参照。Transformation of host cells, preparation of DNA from host cells, etc. can be carried out according to known conventional methods (Ilinnen et al.
et al., Proc, Natl, ^cad, sci, UsA
751929 (1978) and Ito et al., J. Bac.
See Teriol, 153165 (1983).
本発明の発現ベクターを用いて外来遺伝子の発現を行う
には、例えば、外来遺伝子を含むDNA断片を、必要な
らばT4DNAポリメラーゼ等を用いて平滑末端とした
後、Sma Iで切断した発現ベクターに連結し、得ら
れた組換えプラスミドをキャンディダ・マルトーサCH
1株に導入すれば良い。To express a foreign gene using the expression vector of the present invention, for example, a DNA fragment containing the foreign gene is made blunt-ended using T4 DNA polymerase, etc., if necessary, and then inserted into an expression vector cut with Sma I. The resulting recombinant plasmid was ligated to Candida maltosa CH.
It is sufficient to introduce it into one stock.
外来遺伝子産物が生産されているか否かについては、生
化学分野で公知の常法、例えば当該遺伝子産物に対する
抗体との結合反応等を用いて確認する事ができる。Whether or not a foreign gene product is produced can be confirmed using a conventional method known in the field of biochemistry, such as a binding reaction with an antibody against the gene product.
次に本発明を、実施例をもって具体的に説明するが、実
施例は具体的な認識を得る一助としてのみ挙げられてい
るものであり、特許請求の範囲を何ら限定するものでは
ない。Next, the present invention will be explained in detail with reference to examples, but the examples are given only to help give a concrete understanding, and do not limit the scope of the claims in any way.
実施例1;プロモーター領域i域を含むDNA 片の
取得
キャンディダ・マルトーサ(Candida malt
osa)IAM12247株(野性株)をYEPD培地
(酵母エキス1%、ペプトン2%、グルコース2%)で
30’C148時間培養した後、収菌し、染色体DNA
を抽出した。Example 1: Obtaining a DNA fragment containing promoter region i region Candida maltosa (Candida maltosa)
osa) IAM12247 strain (wild strain) was cultured in YEPD medium (yeast extract 1%, peptone 2%, glucose 2%) for 30'C148 hours, harvested, and the chromosomal DNA
was extracted.
コノ染色体DNA約10u、を緩衝液A(10mM T
ris ・IIcI pH”1.5.100mM Na
C1,10mM MgCIz、la+M DTT)及び
20単位の5au3ΔIを含む50μlの反応液中で3
7°C15分間反応させて染色体のDNAの切断を行っ
た後、0%〜10%のショ糖密度勾配遠心(日立RPS
410−ター、28.0OOrp+w 、22時間)を
行って約1〜2KbのDNA断片混合物を回収した。一
方、プラスミドpPLCI (第2図)約10μgを前
記の緩衝液A及び24単位のBamHIを含む50μl
の反応液中で30°C118時間インキュベートした後
、フェノール抽出、エタノール沈澱を行ってDNAを回
収した。Approximately 10 u of Konochromosomal DNA was added to buffer A (10 mM T
ris・IIcI pH”1.5.100mM Na
C1, 10mM MgCIz, la+M DTT) and 20 units of 5au3ΔI in a 50μl reaction.
After reacting at 7°C for 15 minutes to cut the chromosomal DNA, 0% to 10% sucrose density gradient centrifugation (Hitachi RPS
410-ter, 28.0 OOrp+w, 22 hours) to recover a mixture of DNA fragments of about 1 to 2 Kb. Meanwhile, about 10 μg of plasmid pPLCI (Figure 2) was added to 50 μl containing the above buffer A and 24 units of BamHI.
After incubating in the reaction solution at 30°C for 118 hours, phenol extraction and ethanol precipitation were performed to recover the DNA.
このDNA約0. 1μgと上記の約1〜2Kbの染色
体DNA断片混合物0.1μgを緩衝液B (20mM
Tris、IICI(p)17.6)、1kM Mg
Crt、10m)IDTT 、 1mHATP)及び3
50単位のT4 DNAリガーゼを含むlOμ2の反
応液中で12°C,18時間反応させて連結した。This DNA is approximately 0. 1 μg and 0.1 μg of the above approximately 1-2 Kb chromosomal DNA fragment mixture were added to buffer B (20 mM
Tris, IICI (p) 17.6), 1kM Mg
Crt, 10m) IDTT, 1mHATP) and 3
The ligation was performed by reacting at 12°C for 18 hours in a 10μ2 reaction solution containing 50 units of T4 DNA ligase.
この反応液を用いて大腸菌IE、coli JA221
株を形質転換し、得られたアンピシリン耐性の形質
転換体のコロニーよりプラスミドを分画しキャンディダ
・マルトーサのシーンライブラリーとした。Using this reaction solution, E. coli IE, coli JA221
The strain was transformed, and plasmids were fractionated from colonies of the obtained ampicillin-resistant transformants and used as a Candida maltosa scene library.
次にキャンディダ・マルトーサ(Candjda ma
ltos紋J288株(ロイシン要求性株、FERM
P〜9478 、特願昭62−198962)を宿主と
してItoらの、リチウム金属法を用いて、上記のシー
ンライブラリーで形質転換し、グルコース又はグリセロ
ールもしくはテトラデカン2%を含むSD培地(組成を
表1に示す)のプレート上で30”C110日間生育さ
せた。次に、各々の炭素源で生育してきた形質転換体を
上記の炭素源2%を含む最少培地(&Il成を表2に示
す)のプレート上で生育させた所、表3に示される様に
、いずれの炭素源でも生育する形質転換体(A群)及び
グルコースでは生育しないがグリセロール及びテトラデ
カンで生育する形質転換体(B群)が得られた。Next, Candida Maltosa (Candjda ma
ltos Crest J288 strain (leucine auxotrophic strain, FERM
P~9478, Japanese Patent Application No. 62-198962) was transformed with the above scene library using the lithium metal method of Ito et al. The transformants grown on each carbon source were grown on a 30" plate (shown in Table 1) for 110 days. Next, the transformants grown on each carbon source were grown on a minimal medium containing 2% of the above carbon source (the composition is shown in Table 2). As shown in Table 3, transformants that grew on any carbon source (group A) and transformants that did not grow on glucose but grew on glycerol and tetradecane (group B). was gotten.
A群及びB群の形質転換体により各々1株ずつを選び、
プラスミドを調整して、制限酵素間装パターンの解析を
行った所、第1図に示される様な挿入DNA断片を有す
るプラスミドρCP−AI及びpCC−81が得られた
。 pCP−AI及びpCP−81のプロモーター領
域を含む挿入DNA断片のDNA塩基配列をDideo
Ky法を用いて決定した所、各々第5図及び第6図に示
される様な塩基配列であった。Select one strain each from group A and group B transformants,
When the plasmids were prepared and the restriction enzyme intercalation pattern was analyzed, plasmids ρCP-AI and pCC-81 having inserted DNA fragments as shown in FIG. 1 were obtained. The DNA base sequences of the inserted DNA fragments containing the promoter regions of pCP-AI and pCP-81 were
As determined using the Ky method, the base sequences were as shown in FIGS. 5 and 6, respectively.
表1.CD培地の組成
表2.最少培地の組成
表3.最小培地上で生育する形質転換株+ 生育
生育せず
実施例2:
朋
ベク
V=へ1び
B1の
プラスミドpCP−AI約5μgを前記の緩衝液A及び
20単位のl1ae IIを含む50μ2の反応液中で
、またプラスミドpCP−81約5μgを前記の緩衝液
A、20単位のl1ae11及び20単位のBgl I
を含む50μ!の反応液中で37°C,18時間インキ
ユベートシた後、0.6%アガロースゲル電気泳動を行
って、プロモーター領域を含む約0. 9Kb及び約2
. OKbのDNA断片を得た。Table 1. Composition table 2 of CD medium. Composition table 3 of minimal medium. Transformed strain growing on minimal medium + growth Not growing Example 2: Approximately 5 μg of the plasmid pCP-AI of Homobek V = He1 and B1 was added to a 50 μ2 reaction containing the above buffer A and 20 units of l1ae II. Approximately 5 μg of plasmid pCP-81 was also mixed in the buffer A described above, 20 units of l1ae11 and 20 units of Bgl I.
50μ including! After incubation at 37°C for 18 hours in a reaction solution of 0.6% agarose gel electrophoresis, approximately 0.6% containing the promoter region was incubated. 9Kb and about 2
.. A DNA fragment of OKb was obtained.
このDNA断片を、67mM Tris ・IIcI
(pH8,8) 。This DNA fragment was treated with 67mM Tris.IIcI.
(pH 8,8).
6.7 mM MgCb、 16.6mM (NH4
)zsO4110mM 2−メルカプトエタノール、6
.7μ門EDT^、0.0167%ウシ血清アルブミン
、2IIIM dNTT’(dATP、dTTP、dG
TP、dCTP)、及び3単位の74 DNAポリメラ
ーゼを含む20μlの反応液中で37°C55分間イン
キユヘートした後、フェノール抽出、エタノール沈澱を
行ってDNAを回収した。6.7mM MgCb, 16.6mM (NH4
) zsO4110mM 2-mercaptoethanol, 6
.. 7 μm EDT^, 0.0167% bovine serum albumin, 2IIIM dNTT' (dATP, dTTP, dG
After incubation at 37°C for 55 minutes in a 20 μl reaction solution containing 3 units of 74 DNA polymerase, phenol extraction and ethanol precipitation, the DNA was recovered.
さらに、回収したDNA断片とすでにリン酸化されてい
る市販のBag旧リンカ−(約0,02 Azb。Furthermore, the recovered DNA fragments were combined with a commercially available Bag old linker (approximately 0.02 Azb) that had already been phosphorylated.
単位)を、前記の緩衝液B及び350単位のT4DNA
リガーゼを含む10μlの反応液中で12°C218時
間反応させて連結した後、フェノール抽出、エタノール
沈澱を行ってDNAを回収した。units) from the above buffer B and 350 units of T4 DNA.
After ligating by reacting for 218 hours at 12°C in a 10 μl reaction solution containing ligase, the DNA was recovered by phenol extraction and ethanol precipitation.
一方、プラスミドpUc19Es約4μgを前記の緩衝
液A及び20単位のBaIIIIllを含む20μ2の
反応液中で30°C118時間インキエベートした後、
フェノール抽出、エタノール沈澱を行ってDNAを回収
した。On the other hand, about 4 μg of plasmid pUc19Es was incubated in a 20 μ2 reaction solution containing the above buffer A and 20 units of BaIIIll at 30° C. for 118 hours.
DNA was recovered by phenol extraction and ethanol precipitation.
このDNA約0.IIIgと前記のプロモーター領域を
含む約0.9 Kb及び約2.OKbのDNA断片各々
約0.1μgを前記の緩衝液B及び350単位のT4D
NAリガーゼを含む10μlの反応液中で12°C,1
8時間反応させて連結した。This DNA is approximately 0. approximately 0.9 Kb and approximately 2.0 Kb containing IIIg and the promoter region described above. Approximately 0.1 μg of each OKb DNA fragment was added to the above buffer B and 350 units of T4D.
12°C in a 10 μl reaction solution containing NA ligase.
The mixture was reacted for 8 hours and ligated.
次に、この反応液を用いて大腸菌旦、製旦 JA221
株を形質転換し、アンピシリン耐性の形質転換体を得た
。Next, using this reaction solution, Escherichia coli and Seitan JA221
The strain was transformed and ampicillin-resistant transformants were obtained.
それらの形質転換体よりプラスミドDNAを調製し、制
限酵素間然パターンの解析を行って第3図に示される様
なプラスミドPUCP−AI及びpucp−[11を含
む形質転換体を選び出した。Plasmid DNA was prepared from these transformants, and the restriction enzyme pattern was analyzed to select transformants containing plasmids PUCP-AI and pucp-[11 as shown in FIG. 3.
プラスミドpUcP−AI及びpUcP−Bl各々約1
0μgを前記の緩衝液A及び24単位のBstEIIを
含む50μ2の反応液中で60°C11時間後インキユ
ヘートした後、フェノール抽出、エタノール沈澱を行っ
てDNAを回収した。さらに前記の緩衝液A及び20単
位のPstlを含む50μlの反応液中で37°C11
8時間インキュベートした後フェノール抽出、エタノー
ル沈澱を行ってDNAを回収した。Plasmids pUcP-AI and pUcP-Bl each about 1
After incubating 0 μg in a 50 μ2 reaction solution containing the above-mentioned buffer A and 24 units of BstEII at 60° C. for 11 hours, phenol extraction and ethanol precipitation were performed to recover DNA. Further, in a 50 μl reaction solution containing the above buffer A and 20 units of Pstl at 37° C.
After incubation for 8 hours, phenol extraction and ethanol precipitation were performed to recover DNA.
このDNAを宝酒造製K11o−5equence用D
eletionKiLを用いて25℃で反応させ、30
秒ごとにサンプリングしてDNAを回収した。回収した
DNA約0.5μgとすでにリン酸化されている市販の
Sea Iリンカ−を、前記の緩衝液B及び350単位
の74DNAリガーゼを含む10μPの反応液中で12
°C11日時間反応させて連結した。This DNA was converted into Takara Shuzo K11o-5equence D.
React at 25°C using eletionKiL and incubate for 30
DNA was collected by sampling every second. Approximately 0.5 μg of the recovered DNA and the commercially available Sea I linker, which had already been phosphorylated, were incubated for 12 hours in a 10 μP reaction solution containing the above buffer B and 350 units of 74 DNA ligase.
The mixture was reacted at °C for 11 days and ligated.
次に、この反応液を用いて大腸菌E、coli JA
221株を形質転換し、アンピシリン耐性の形質転換体
を得た。Next, using this reaction solution, E. coli E, coli JA
221 strain was transformed to obtain ampicillin-resistant transformants.
それらの形質転換体よりプラスミドDNAを調製し、5
LIla■切断部位近傍のDNA塩基配列を決定して、
LEU2遺伝子領域のATCまでが欠失した第3図に示
される様なプラスミドpUcP−^1d及びpUCP−
Bldを含む形質転換体を選び出した。Plasmid DNA was prepared from these transformants, and
Determine the DNA base sequence near the LIla■ cleavage site,
Plasmids pUcP-^1d and pUCP- as shown in Fig. 3, which are deleted up to ATC in the LEU2 gene region
Transformants containing Bld were selected.
プラスミドpUcP−Ald及びpUcP−Bid各々
約IOμgを前記の緩衝液A、20中20単
位のSma lを含む5Oagの反応液中で37°C,
18時間インキユヘートした後、0.6%アガロー
スゲル電気泳動を行って、プロモーター領域を含む約0
.6Kb.及び約1.7KbのDNA断片を得た。Approximately IO μg of each of plasmids pUcP-Ald and pUcP-Bid were incubated at 37°C in a 5Oag reaction solution containing 20 units of SmaI in 20% of the buffer A described above.
After incubation for 18 hours, 0.6% agarose gel electrophoresis was performed to detect approximately 0.0
.. 6Kb. A DNA fragment of approximately 1.7 Kb was obtained.
一方、プラスミドpUcT1約5μgを前記の緩衝?&
A、20単位のOam III及び20単位のSma
+を含む50μ2の反応液中で37°C1■8時間イ
ンキュベートした後、フェノール抽出、エタノール沈澱
を行って、ターミネータ−領域を含むプラスミドを回収
した。このDNA約0.1μgと前記プロモーター領域
を含む約1.7KbのDNA断片各々約0.1μgを前
記の緩衝液B及び350単位のT4DNAリガーゼを含
む1Oagの反応液中で12°C1■8時間反応させて
連結した。On the other hand, approximately 5 μg of plasmid pUcT1 was added to the buffer described above. &
A, 20 units of Oam III and 20 units of Sma
After incubation at 37°C for 18 hours in a 50μ2 reaction solution containing +, phenol extraction and ethanol precipitation were performed to recover the plasmid containing the terminator region. Approximately 0.1 μg of this DNA and approximately 0.1 μg each of the approximately 1.7 Kb DNA fragment containing the promoter region were placed in a 1 Oag reaction solution containing the above buffer B and 350 units of T4 DNA ligase at 12°C for 18 hours. They were reacted and ligated.
次にこの反応液を用いて大腸菌E, coli JA
221株を形質転換し、アンピンリン耐性の形質転換
体を得た。Next, using this reaction solution, E. coli E, coli JA
221 strain was transformed, and a transformant resistant to ampinrin was obtained.
これらの形質転換体よりプラスミドDNAを調製し、制
限酵素開裂パターンの解析を行って、第3図に示される
様なプラスミドpSC−Al及びpSC−Blを含む形
質転換体を選び出した。Plasmid DNA was prepared from these transformants, and the restriction enzyme cleavage pattern was analyzed to select transformants containing plasmids pSC-Al and pSC-Bl as shown in FIG.
プラスミドpSC−AI及びpSC−Bl各々約10μ
gを前記の11街液A、20単位のBa+m旧及び20
単位の5allを含む50μ2の反応液中で37°c1
18時間インキエベートした後、0.6%アガロース
ゲル電気泳動を行って、プロモーター領域及びターミネ
ータ−領域を含む約0.9Kb及び約2.OKbのDN
A断片を得た。このDNA断片を、50mM Tris
・IIcI(pt17.2)、 10n+M Mg5O
a, 0.1mM DTT, 50 1テg/ml B
SA, 0.2mM dNTP(dATl”、dTTP
,dGTP,dCTP)及び3、5単位のDNAポリメ
ラーゼ(Klenow Fragment)を含む25
alの反応液中で22°C130分間インキュベートし
た後、フェノール抽出、エタノール沈澱を行ってDNA
を回収した。回収したDNAとすでにリン酸化されてい
る市販のBgl IIリンカ−を、前記の緩衝液B及び
350単位のT4DNAリガーゼを含むlθμ2の反応
液中で12°C1■8時間反応させて連結した後、フェ
ノール抽出、エタノール沈澱を行ってDNAを回収した
。Plasmids pSC-AI and pSC-Bl each about 10μ
g to the above 11 street solution A, 20 units of Ba+m old and 20
37°c1 in a 50μ2 reaction solution containing 5all units of
After incubation for 18 hours, 0.6% agarose gel electrophoresis was performed to obtain approximately 0.9 Kb and approximately 2.0 Kb containing promoter and terminator regions. OKb's DN
Fragment A was obtained. This DNA fragment was treated with 50mM Tris.
・IIcI (pt17.2), 10n+M Mg5O
a, 0.1mM DTT, 50 1teg/ml B
SA, 0.2mM dNTP (dATl”, dTTP
, dGTP, dCTP) and 3.5 units of DNA polymerase (Klenow Fragment).
After incubating for 130 minutes at 22°C in the reaction solution of al., the DNA was extracted with phenol and precipitated with ethanol.
was recovered. The recovered DNA and a commercially available Bgl II linker that had already been phosphorylated were ligated by reacting for 18 hours at 12°C in a 1θμ2 reaction solution containing the aforementioned buffer B and 350 units of T4 DNA ligase. DNA was recovered by phenol extraction and ethanol precipitation.
一方、プラスミドp[1THIOB約5μgを前記の緩
衝液A及び20単位のBglIIを含む50μlの反応
液中で37°C.to時間インキュベートした後、フェ
ノール抽出、エタノール沈澱を1テっでDNAを回収し
た。Separately, about 5 μg of plasmid p[1THIOB was incubated at 37°C in a 50 μl reaction solution containing the above buffer A and 20 units of BglII. After incubation for up to hours, DNA was recovered by phenol extraction and ethanol precipitation.
このDNA約O0lμgと前記のプロモーター及びター
ミネータ−を含む約0.9Kb及び約2.OKbのDN
A断片各々約0.1μgを前記の緩衝液B及び350単
位の74 DNAリガーゼを含む10uNの反応液中
で12°C5■8時間反応させて連結した0次にこの反
応液を用いて大腸[]E,coli JA221株を
形質転換し、アンピシリン耐性の形質転換体を得た。Approximately 0.9 Kb and approximately 2.0 μg of this DNA and approximately 0.9 Kb including the promoter and terminator described above. OKb's DN
Approximately 0.1 μg of each A fragment was ligated by reacting for 58 hours at 12°C in a 10 uN reaction solution containing the above buffer B and 350 units of 74 DNA ligase.Next, this reaction solution was used to inject into the large intestine [ ] E. coli strain JA221 was transformed to obtain ampicillin-resistant transformants.
こちらの形質転換体よりプラスミドDNAを調製し、制
限酵素開裂パターンの解析を行って図面4に示されてる
様なプラスミドpCEV−AI及びρC1’V− B1
を含む形質転換体を選び出した。Plasmid DNA was prepared from this transformant, and the restriction enzyme cleavage pattern was analyzed to generate plasmids pCEV-AI and ρC1'V-B1 as shown in Figure 4.
A transformant containing the following was selected.
第1図は、pCP−AI及びρCP−Blに挿入されて
いる、キャンディダ・マルトーサ染色体DNA由来のプ
ロモーター領域を含むDNA断片を示す。
第2図は、プロモーター検索用プラスミドベクタ−
pPLclを示す。
第3図は、ρ5C−AI及びpSC−Blの構築過程を
示す。
第4図は、発現ベクターpCEV−AI及びpc[!V
−81の構築過程を示す。
第5図は、pCP−AIに含まれるプロモーター領域の
DNA塩基配列を示す。
第6図は、pCP−81に含まれるプロモーター領域の
DNA塩基配列を示す。
第1図
pCP−AI H−一一一←0.6KbpSau3.A
I 5au3AI調べた制限酵素
EcoRI、 PstI、 Xho I、 C1aI、
BamHI、 Smal、 Hindll第8図
第2図
BamHI
第4図
第5図
pCP−AIプロモーター領域D N A配列第6図
pcp −B lプロモーター領域DNA配列(その1
)+0 20 30
40 +50GATCT
GTCAT CAAAGGAAAT ACGACGAA
Tr CCGACGACCA TGATTT、ATGT
GATCATrAGCCTCTrTCGCA GGAC
TACTCA ACAATCCACCATrCTTCA
CCTATCAATCTA TATrTAGTACGT
rCTCGGCG TGTCTrTrCA AACAA
AA人人人CCATCATC人A TGTCTrCAT
CTrCTTCTGTCGTrGTTGATA GT
TT’TCTCTTTTTc人ACAAT TGCG
CGGCACGATACGCTCT TGATGAA
ATr CAACAGATCT+10
120 130 140
150AAAGAAATAA AAAAG
C人ATT TrCAAATrTr TGCTATT
CCA GATrAAGATGGATAAATAGT
TTAAAGTrCG CCGATCTCCG
人TCCCCTCTG GTCCGCCCAT16
0 +70 +80
190 200TTrGATr
ACT AAATr、AGTTr AAAC人AT思G
んり請G人人TCGTGCT口TCCAAATACAT
AA GCCCAGC人AA AAATrTGAAA
GAAAAGGTrT CTGTATCATA2
+0 220 230
240 250cTTTTT人T
CA CGTGTGTTGA TrCTCATCC
T TACATAT!TT GCAACTrTGG
ATrATrATrT GCATrTTGAT G
TAAATCTGT TTrATTG人AG AA
TGATTTAGTTGAACTGAT TTrATC
AAGA AGCTGGTGGA TAATAATCT
CAGGTrT口TrCCAATCACCCACAAA
GAAGG GGAGAAAluT 人ATAAACC
ACAGATATmTTTTCTrTATr TCT
TGCGTrT GAGA人ATCAT GG人人
AATCTr TTCACC人人人人wcTTTcT
rr rr口TrTCCT TCCTCGCAGT T
ATGTGTGTCGCGATCATCATCATrA
CATCAATrGTrG7CCGACTATGGT
AGTCA/ITrGT TTGTTrTTGGCGT
TTTTTAT TTTTAACAGG TCCTCC
TAAA ん1ゴ7rrr’rr TTTGGTTTG
AGAAGTGGTGT G人ATGAGAAA
AGAAGAA’lAA TrTATCGGGA
TAGA、ATATTTTTTrACCGAT TCC
AGAT、ACA ACA、TCAAATA AATC
TAACTG GGATrTrTCTCGCGTCT]
”CG CGAATCTrTr TTTTCGAAC
T AAAGT人TTGT TrTrTrACTA
TTTrTGCTTA CAACCTATrT CTT
TTCTTTT TATTTTGTTT TTTGTr
ACAGTATAGAAATCACTCGTAAACA
ACAAAACAA AG、リリ訪人TAA CACT
AAAG人人ACAACGC人AA CCCTAAC
ATT CCAATGTGGA GAAGAATC
AA AACT7ACAGAA工C
650、670680690700
TATCACT、AAG ACAGACAAACAAA
CAATTT=へ cccc、起へ人ACA んへOん
−へG人TC第6図
(その2)
TAATrGTNGT CTAGTrTTAT T
CTr’GTATCG CACACATCACTAC
ATCAATACGmTGTCA CCGCTrTrC
T GCTCCGGTACTrGTCCCCGA AA
TAGGGATTTCTrTTITAT TAGTAC
AATCTAGTCATAAT AGATATGGGG
AAAATAATATTATrATmT AATAG
TmT ATrTTCCC人T CACGCT人A
GCAAAATAAATAAAAAACGCCG TT
GTTCTGCT GATGCTTTI’T TrTC
TrTr’CT TrCCATrTrACGATrCA
AACTrTGGTrCTT ATCTGACATCG
ACCCAGGTr TTCTrTITrT+010
1020 1030
+040 1050TrTrCTTGAA
TCTTGTrATCAACAATATAA CGA
TGAAGAA GAAAAATn丁GCCCTCTC
GT 人TrTCC人ATA ACTTATCTCT
AGGTrTAACA CGTGTrOTTc11
30 ]+40 1+50C
GCCATrCTA AACGGAAAACTCAA
TrCATCTCACTCAATr 人AAG人T人
TAG]480 1190
1200CCTTACCTrTCTrTCCCCAG
CCCGTGAAAA TATrTCGATr ATC
TGGCGAA+230 1240
+250TAGTACTAGG AGAAA
CTCACCTGTrTAIIAAATCAAAAAT
CG入んυ訪スATG1280 +290
1300CCGTATGTGT ACん
り仏んり請人AGんリリ誂TA GTCCACTA1了
CCCCACんりA+330 1340
1350MACAATAT TrTCTAGA
AA GCGTr、AACAC】360
ATCTTGATCFIG. 1 shows a DNA fragment containing a promoter region derived from Candida maltosa chromosomal DNA that has been inserted into pCP-AI and ρCP-Bl. Figure 2 shows plasmid vectors for promoter search.
pPLcl is shown. FIG. 3 shows the construction process of ρ5C-AI and pSC-Bl. FIG. 4 shows expression vectors pCEV-AI and pc[! V
-81 construction process is shown. FIG. 5 shows the DNA base sequence of the promoter region contained in pCP-AI. FIG. 6 shows the DNA base sequence of the promoter region contained in pCP-81. Figure 1 pCP-AI H-111←0.6KbpSau3. A
Restriction enzymes tested by I5au3AI: EcoRI, PstI, XhoI, C1aI,
BamHI, Smal, HindllFigure 8Figure 2BamHI Figure 4Figure 5 pCP-AI promoter region DNA sequenceFigure 6 pcp-Bl promoter region DNA sequence (Part 1)
)+0 20 30
40 +50 GATCT
GTCAT CAAAGGAAAT ACGACGAA
Tr CCGACGACCA TGATTT, ATGT
GATCATrAGCCTCTrTCGCA GGAC
TACTCA ACAATCCACCATTrCTTCA
CCTATCAATCTATATrTAGTACGT
rCTCGGCG TGTCTrTrCA AACAA
AA person person CCATCATC person A TGTCTrCAT
CTrCTTCTGTCGTrGTTGATA GT
TT'TCTCTTTTTTc人ACAAT TGCG
CGGCACGATACGCTCTTGATGAA
ATr CAACAGATCT+10
120 130 140
150AAAGAAAATAAAAAAG
C person ATT TrCAAATrTr TGCTATT
CCA GATrAAGATGGATAAAATAGT
TTAAAGTrCG CCGATCTCCG
PersonTCCCCTCTG GTCCGCCCAT16
0 +70 +80
190 200TTrGATr
ACT AAATr, AGTTr AAAC person AT thought
NRIBUG 人人TCGTGCT口TCCAAATACAT
AA GCCCAGC人AA AAATrTGAAA
GAAAAGGTrT CTGTATCATA2
+0 220 230
240 250cTTTTTT person T
CA CGTGTGTTGA TrCTCATCC
T TACATAT! TT GCAAACTrTGG
ATrATrATrT GCATrTTGAT G
TAAATCTGT TTrATTG person AG AA
TGATTTAGTTGAACTGAT TTrATC
AAGA AGCTGGTGGA TAATAATCT
CAGGTrT-TrCCAATCACCCACAAA
GAAGG GGAGAAAluT person ATAAAACC
ACAGATATmTTTTTCTrTATr TCT
TGCGTrT GAGA人ATCAT GG人人AATCTr TTCACC人人人人wcTTTcT
rr rrmouth TrTCCT TCCTCGCAGT T
ATGTGTGTCGCGATCATCATCATTrA
CATCAATrGTrG7CCGACTATGGT
AGTCA/ITrGT TTGTTrTTGGCGT
TTTTTAT TTTTAACAGG TCCTCC
TAAA n1go7rrr'rr TTTGGTTTG
AGAAGTGGTGT G person ATGAGAAA
AGAAGAA'lAA TrTATCGGGA
TAGA, ATATTTTTTTrACCGAT TCC
AGAT, ACA ACA, TCAAATA AATC
TAACTG GGATrTrTCTCGCGTCT]
”CG CGAATCTrTr TTTTCGAAC
TAAAGT人TTGT TrTrTrACTA
TTTrTGCTTA CAACCTATrT CTT
TTCTTTT TATTTTGTTT TTTGTr
ACAGTATAGAAATCACTCGTAAACA
ACAAAACAA AG, LILI VISIT TAA CACT
AAAG person ACAACGC person AA CCCTAAC
ATT CCAATGTGGAGAAGAATC
AA AACT7ACAGAA C 650, 670680690700 TATCACT, AAG ACAGACAAAACAAA
CAATTT=to cccc, rise to person ACA Nhe On-to G person TC Figure 6 (Part 2) TAATrGTNGT CTAGTrTTAT T
CTr'GTATCG CACACATCACTAC
ATCAATACGmTGTCA CCGCTrTrC
T GCTCCGGTACTTrGTCCCCGA AA
TAGGATTTCTrTTITAT TAGTAC
AATCTAGTCATAAT AGATATGGGGG
AAAATAATATATTATTrATmT AATAG
TmT ATrTTCCC person T CACGCT person A
GCAAAATAAAAATAAAAACGCCG TT
GTTCTGCT GATGCTTTI'T TrTC
TrTr'CT TrCCATrTrACGATrCA
AACTrTGGTrCTT ATCTGACATCG
ACCCAGGTr TTCTrTITrT+010
1020 1030
+040 1050TrTrCTTGAA
TCTTGTrATCAACAACATATAA CGA
TGAAGAA GAAAAAATn Ding GCCCTCTC
GT person TrTCC person ATA ACTTATCTCT
AGGTrTAACA CGTGTrOTTc11
30]+40 1+50C
GCCATrCTA AACGGAAAACTCAA
TrCATCTCACTCAATr Person AAG Person T Person TAG] 480 1190
1200CCTTACCTrTCTrTCCCAG
CCCGTGAAAA TATrTCGATr ATC
TGGCGAA+230 1240
+250TAGTACTAGGAGAAA
CTCACCTGTrTAIIIAAATCAAAAAAT
CG entered υ visit ATG1280 +290
1300CCGTATGTGT AC Nributsurenribenjin AGnlili customization TA GTCCACTA1 completedCCCCACnriA+330 1340
1350MACAATAT TrTCTAGA
AA GCGTr, AACAC】360 ATCTTGATC
Claims (5)
dida¥¥maltosa¥)の細胞内において機能
する複製開始点、該酵母染色体DNA由来のプロモータ
ー領域、プロモーター領域下流に設けられた制限酵素切
断部位及びサッカロマイセス・セレビシエ(Sacch
aromyces¥¥cerevisiae¥)のLE
U2遺伝子由来のターミネーター領域を含有する事を特
徴とする発現ベクター(1) Non-spore yeast Candida maltosa (¥Can
a replication origin that functions in cells of Saccharomyces cerevisiae (Saccharomyces cerevisiae), a promoter region derived from the yeast chromosomal DNA, a restriction enzyme cleavage site provided downstream of the promoter region.
LE of aromyces¥¥cerevisiae¥)
An expression vector characterized by containing a terminator region derived from the U2 gene
列が下記の配列である特許請求の範囲第1項記載の発現
ベクター 【遺伝子配列があります】(2) The expression vector according to claim 1, wherein the nucleotide sequence of the DNA containing the promoter region is the following sequence [there is a gene sequence]
第2項記載の発現ベクター(3) The expression vector according to claim 2, which is plasmid pCEV-A1.
列が下記の配列である特許請求の範囲第1項記載の発現
ベクター 【遺伝子配列があります】(4) The expression vector according to claim 1, wherein the nucleotide sequence of the DNA containing the promoter region is the following sequence [there is a gene sequence]
第4項記載の発現ベクター。(5) The expression vector according to claim 4, which is plasmid pCEV-B1.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22594788A JPH0272882A (en) | 1988-09-08 | 1988-09-08 | Expression vector |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP22594788A JPH0272882A (en) | 1988-09-08 | 1988-09-08 | Expression vector |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH0272882A true JPH0272882A (en) | 1990-03-13 |
Family
ID=16837387
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP22594788A Pending JPH0272882A (en) | 1988-09-08 | 1988-09-08 | Expression vector |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH0272882A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014039533A (en) * | 2012-03-15 | 2014-03-06 | Toyota Central R&D Labs Inc | Production method of expression product of exogenous gene in yeast, expression control agent in yeast and its use |
-
1988
- 1988-09-08 JP JP22594788A patent/JPH0272882A/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014039533A (en) * | 2012-03-15 | 2014-03-06 | Toyota Central R&D Labs Inc | Production method of expression product of exogenous gene in yeast, expression control agent in yeast and its use |
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