JPH0568582A - Method for treating culture solution for secreting and producing hirudins - Google Patents
Method for treating culture solution for secreting and producing hirudinsInfo
- Publication number
- JPH0568582A JPH0568582A JP25701291A JP25701291A JPH0568582A JP H0568582 A JPH0568582 A JP H0568582A JP 25701291 A JP25701291 A JP 25701291A JP 25701291 A JP25701291 A JP 25701291A JP H0568582 A JPH0568582 A JP H0568582A
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- Prior art keywords
- culture
- hirudins
- culture solution
- hirudin
- producing
- Prior art date
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【０００１】[0001]
【発明の属する分野】本発明は、ヒルジン類生産菌培養
液、特に遺伝子組換えヒルジン生産大腸菌培養液からヒ
ルジン類を採取するための培養液の処理方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for treating hirudin-producing bacterium culture broth, particularly for treating hirudin from a recombinant recombinant hirudin-producing Escherichia coli broth.
【０００２】[0002]
【従来の技術】現在ヒルジン類の分泌生産は、酵母、枯
草菌、大腸菌等の遺伝子組換え微生物を用いて行なわれ
ている、これらのヒルジン類の生産に関する特許文献、
あるいは技術文献は数多くみられるが、これらの方法
は、一般的にはこれらの菌体を培養し、培養終了後、培
養液から菌体を除去殺菌し、培養上清からヒルジン類を
単離精製している。これらの文献には、培養液から菌体
を除去する前に、培養液を殺菌することについては記載
されていない。2. Description of the Related Art Currently, secretory production of hirudins is carried out using genetically modified microorganisms such as yeast, Bacillus subtilis, Escherichia coli, etc. Patent documents relating to the production of these hirudins,
Alternatively, although there are many technical literatures, these methods generally involve culturing these bacterial cells, removing the bacterial cells from the culture solution and sterilizing after completion of the culture, and isolating and purifying hirudins from the culture supernatant. is doing. These documents do not describe the sterilization of the culture solution before removing the bacterial cells from the culture solution.
【０００３】この菌体の除去手段は、遠心分離、膜濾過
等が一般的である。しかし、組換え微生物であるので、
できるだけ早い段階での殺菌及び充分な除菌が必要であ
る。特にこのことは、大規模にヒルジン類を生産するス
ケールになると不可欠である。大腸菌の大スケール培養
の場合、遠心分離のみでは除菌は十分ではなく、フィル
ター等による濾過が必要である。しかし、このフィルタ
ーによる濾過はフィルターがすぐ目づまりを起こす欠点
がある。[0003] Centrifugation, membrane filtration, etc. are generally used as means for removing the cells. However, because it is a recombinant microorganism,
Sterilization and sufficient sterilization at the earliest stage are necessary. In particular, this is indispensable at the scale for producing hirudins on a large scale. In the case of large-scale culture of Escherichia coli, centrifugation alone is not sufficient for eradication, and filtration with a filter or the like is necessary. However, the filtration with this filter has a drawback that the filter is immediately clogged.
【０００４】又、組換え大腸菌によるペプチド生産では
大腸菌のプロテアーゼによる生産物の分解が問題となる
(K.Kitano J. of Biotechnology 5 77, 1987)。これは
ヒルジンの分泌生産においても同様で、特に培地中にヒ
ルジンが多く蓄積される培養後期にヒルジンが分解さ
れ、その蓄積量が減少してしまうことの原因であると考
えられる。In the production of peptides by recombinant Escherichia coli, degradation of the products by E. coli protease poses a problem.
(K. Kitano J. of Biotechnology 5 77, 1987). This is also the case in the secretory production of hirudin, and it is considered that this is a cause of the fact that hirudin is decomposed at the latter stage of the culture, where a large amount of hirudin is accumulated in the medium, and the accumulated amount is decreased.
【０００５】除菌後の培養液上清の処理に関しては、ポ
リエチレンイミンで処理し非蛋白を除去し、酢酸でpH４
〜５に酸性化し宿主蛋白を沈澱させる方法（特開平2-13
377)、塩酸でpH３に調整した後70℃で15分間処理し生じ
た沈澱を除去する方法（特開平2-35084)、直接樹脂に吸
着させ疎水クロマトグラフィーを行なう方法（特開平1-
165599）等がある。これらは、いずれも菌体を除いた上
清の処理に関するものである。また、除菌を行なう前に
培養液を直接アンバーライトＸＡＤ−７に吸着させ、菌
体を除く方法（特開平2-13377)もある。しかし、これら
の方法はいずれも殺菌、除菌、ヒルジン分解の防止に関
するものではない。Regarding the treatment of the culture supernatant after sterilization, it is treated with polyethyleneimine to remove nonproteins, and the pH is adjusted to 4 with acetic acid.
-5 to acidify the host protein by precipitation (Japanese Patent Application Laid-Open No. 2-13
377), a method of adjusting the pH to 3 with hydrochloric acid and then treating at 70 ° C. for 15 minutes to remove the formed precipitate (JP-A-2-35084), and a method of directly adsorbing to a resin for hydrophobic chromatography (JP-A-1-35084).
165599) etc. These are all related to the treatment of the supernatant excluding the bacterial cells. There is also a method of adsorbing the culture solution directly to Amberlite XAD-7 before removing the bacteria to remove the bacterial cells (Japanese Patent Laid-Open No. 2-13377). However, none of these methods relates to sterilization, sterilization, or prevention of hirudin decomposition.
【０００６】[0006]
【発明が解決しようとする課題】ヒルジン類の大量生産
を行なう場合、培養終了後の分泌生産菌の殺菌及び除
菌、ヒルジン類の分解の防止さらにその精製を効率的か
つ確実に行なうことが必要である。また、さらにこの方
法がより簡単であることが望ましい。本発明の課題は、
ヒルジン類分泌生産菌、特に組換え大腸菌によるヒルジ
ンの分泌生産において、培養終了後の培養液を処理する
ことによって生産菌の殺菌、菌体の除去、分泌されたヒ
ルジンの分解の防止、不純物蛋白の除去を確実に効率的
に行なうことのできる方法を提供することである。When mass-producing hirudins, it is necessary to sterilize and eradicate secretory-producing bacteria after completion of culture, prevent decomposition of hirudins, and perform purification thereof efficiently and surely. Is. It is also desirable that this method be simpler. The object of the present invention is to
In the secretory production of hirudins by secretory hirudins, especially recombinant Escherichia coli, by treating the culture solution after the culturing, sterilization of the producing bacteria, removal of cells, prevention of the secretion of secreted hirudin, It is to provide a method capable of surely and efficiently performing removal.
【０００７】[0007]
【課題を解決するための手段】すなわち、本発明は、ヒ
ルジン類分泌生産菌培養液の培養終了直後の菌体を含む
培養液を、酸によりpH５以下、好ましくはpH３乃至４に
調整し、これを暫時放置し、その後菌体と培養上清とに
分離することよりなるヒルジン類分泌生産培養液の処理
法に関する。[Means for Solving the Problems] That is, according to the present invention, a culture solution containing cells of a hirudin secretory production bacterium culture medium immediately after the culture is adjusted to pH 5 or less, preferably pH 3 to 4 with an acid. To a treatment method of a hirudin secretory production culture solution, which comprises allowing the cells to stand for a while, and then separating the cells into a culture supernatant and a culture supernatant.
【０００８】本発明におけるヒルジン類分泌生産菌に
は、従来知られている遺伝子組換え手法によってヒルジ
ン類生産性を付与された酵母、枯草菌、大腸菌等が用い
られる。これらは、どのような微生物であってもよい
が、発明の目的からみて遺伝子組換え大腸菌に本発明の
方法を適用することが望ましい。このような大腸菌の例
を挙げると、ヒルジン分泌生産用プラスミドpMTSHVlC３
で形質転換した大腸菌JM109(微工研菌寄3104号）があ
る。As the hirudin secretory producing bacterium in the present invention, yeast, Bacillus subtilis, Escherichia coli etc. to which hirudin productivity has been imparted by a conventionally known gene recombination technique are used. These may be any microorganisms, but it is desirable to apply the method of the present invention to recombinant Escherichia coli for the purpose of the invention. An example of such E. coli is the plasmid pMTSHVlC3 for producing hirudin secretion.
Escherichia coli JM109 (Microtechnical Research Institute No. 3104) transformed with E. coli.
【０００９】培養液についても、従来ヒルジン類分泌生
産菌の培養に用いられている培地であれば、天然培地で
も合成培地でも用いられる。本発明のヒルジン類には、
天然のヒルジンＨＶ−１、ＨＶ−２、ＨＶ−３等或い
は、これらの天然ヒルジンのアミノ酸の一部を置換した
ヒルジン類縁体を挙げることができる。As for the culture medium, either a natural medium or a synthetic medium may be used as long as it is a medium which has been conventionally used for culturing hirudin secretory producing bacteria. The hirudins of the present invention include
Examples include natural hirudin HV-1, HV-2, HV-3 and the like, or hirudin analogs obtained by substituting a part of amino acids of these natural hirudins.
【００１０】本発明では、ヒルジン類分泌生産菌培養液
の培養終了直後の菌体を含む培養液をまず酸によってpH
５以下にする。使用される酸としては、硫酸、塩酸等の
無機酸あるいは酢酸等の有機酸が用いられる。培養液の
pHは５以下に調整するが、特にpH３〜４に調整すると、
ヒルジン活性を低下させることなく、生産菌の死滅率を
高め、その後の濾過のさいのフィルターの目づまりを顕
著に防止することができるのでこのようなpHに調整する
ことが望ましい。また、本発明における培養終了直後
は、培養終了した後直ちに乃至その後数時間程度のヒル
ジン活性が実質的にほとんど低下していない時間を意味
する。次に、pH５以下に調整された菌体を含む培養液を
暫時放置し、その後、菌体と培養上清とに分離する。暫
時放置は４〜３７℃に１０分程度〜数時間放置するもの
であり、また、分離手段としては遠心分離、フィルター
濾過等の手段を単独あるいは併用して用いることができ
る。In the present invention, the pH of the culture solution containing the cells immediately after the completion of the culture of the hirudin secretory production bacterium is first adjusted with an acid.
Set to 5 or less. As the acid used, inorganic acids such as sulfuric acid and hydrochloric acid or organic acids such as acetic acid are used. Of culture
The pH is adjusted to 5 or less, but especially when the pH is adjusted to 3 to 4,
It is desirable to adjust to such a pH, because the killing rate of the producing bacteria can be increased and the clogging of the filter during the subsequent filtration can be significantly prevented without lowering the hirudin activity. Immediately after the completion of the culture in the present invention, it means the time immediately after the completion of the culture or for several hours after that, when the hirudin activity is not substantially reduced. Next, the culture solution containing the cells adjusted to pH 5 or less is left for a while and then separated into the cells and the culture supernatant. Temporary leaving is to leave at 4 to 37 ° C. for about 10 minutes to several hours, and as a separating means, means such as centrifugation and filter filtration can be used alone or in combination.
【００１１】本発明の方法によると、特に、培養終了直
後の菌体を含む培養液を酸によってpH５以下とし、暫時
放置したのでヒルジン類の失活、分解を抑え、菌体の殺
菌を完全に行うことができ、しかも菌体の分離を容易に
する。さらに不純物タンパク質の除去をも可能にするこ
とができる。According to the method of the present invention, the pH of the culture medium containing the cells immediately after the culturing is adjusted to 5 or less with an acid and left for a while so that the inactivation and decomposition of hirudins can be suppressed and the sterilization of the cells can be completed. It can be performed and facilitates the separation of bacterial cells. Furthermore, it is possible to remove impurities proteins.
【００１２】さらに、本発明の特徴について特に大腸菌
を例に挙げて説明する。培養液のpHを酸性あるいはアル
カリ性にすると大腸菌が死滅することは良く知られた事
実であるが、アルカリ性にすると大腸菌のタンパク質が
菌体から溶出することがあり、培養液中のヒルジンの不
純物を増やすことになるので好ましくない。酸性にする
と多くのタンパク質は不溶性となり沈澱するが、ヒルジ
ンは酸性でも溶解性が高く回収率の低下につながらな
い。よって酸性にしたあと遠心分離や濾過等の手段によ
り大腸菌の殺菌と不純物タンパクの除去が可能となる。Further, the features of the present invention will be described by taking Escherichia coli as an example. It is a well-known fact that Escherichia coli is killed when the pH of the culture solution is made acidic or alkaline, but when it is made alkaline, the Escherichia coli protein may elute from the cells and increase the impurities of hirudin in the culture solution. It is not preferable because it will happen. Many proteins become insoluble and precipitate when acidified, but hirudin is highly soluble even in acid and does not lead to a decrease in recovery rate. Therefore, it becomes possible to sterilize Escherichia coli and remove impurity proteins by means such as centrifugation or filtration after acidification.
【００１３】さらに、培養条件によっては大腸菌は異種
タンパクを分解する酵素を生産し、それが培地中のヒル
ジンを分解してしまうことがあるが、その酵素は酸性pH
（pH５以下好ましくは 4.0以下）で失活するので、酵素
による分解を抑えることもできる。又、上記したよう
に、培養液のpHを酸性にすることによりヒルジン以外の
タンパク質が不溶性の沈澱となるが、この生成した沈澱
が大腸菌を除く遠心分離の際にその除菌性能を向上さ
せ、遠心分離後の上清をさらにフィルターで濾過する際
に、その濾過性を向上させる。本発明は、このようにヒ
ルジン類の性質と大腸菌等菌体の性質とを巧みに組合せ
ることによって完成したものである。Furthermore, depending on the culture conditions, Escherichia coli produces an enzyme that decomposes a heterologous protein, which may decompose hirudin in the medium.
Since it is inactivated at a pH of 5 or less, preferably 4.0 or less, it is possible to suppress decomposition by an enzyme. Further, as described above, by making the pH of the culture solution acidic, proteins other than hirudin become insoluble precipitates, but the precipitates formed improve the sterilization performance during centrifugation to remove E. coli, When the supernatant after centrifugation is further filtered with a filter, its filterability is improved. The present invention has been completed by skillfully combining the properties of hirudins with the properties of bacterial cells such as Escherichia coli.
【００１４】次に本発明を実施例を挙げて具体的に説明
する。Next, the present invention will be specifically described with reference to examples.
【実施例１】ヒルジン分泌生産用プラスミドpMTSHVlC３
で形質転換した大腸菌 JM109（特願03-41271微工研菌寄
第3104号）を１ｌの天然培地(Bacto-Tryptone 16g/l,Ba
cto-Yeast extract 10g/l, NaCl 10g/l)中で37℃、18時
間振盪培養し、これを種培養とした。次に19ｌの合成培
地〔(NH4)2SO4 7.1g/l, K2HPO4 1.7g/l,カザミノ酸5g/
l, トリプトファン0.5g/l, グルコース20g/l, MgSO4・7
H20 2.1g/l, CaCl2 0.19g/l, FeCl3 ・6H2O 0.0048g
/l, 塩酸チアミン0.02g/l, H3BO3 4.96 μg/l,(NH4 )6
M07 O24 ・4H2O 0.74μg/l, CuSO4・5H2O 0.5 μg/
l, MnCl2・4H2O 4.3μg/l, ZnSO4・7H20 0.58μg/l 〕
に上記１ｌの種培養液を加えpH7.0,37℃で30ｌ発酵槽を
用いて培養を24時間行なった。12時間の時点でグルコー
ス粉末を 800ｇ加えた。[Example 1] Plasmid pMTSHVlC3 for producing hirudin secretion
Escherichia coli JM109 (Japanese Patent Application No. 03-41271 Microbiology Research Institute No. 3104) transformed with 1 liter of natural medium (Bacto-Tryptone 16g / l, Ba
Cto-Yeast extract 10 g / l, NaCl 10 g / l) was shake-cultured at 37 ° C. for 18 hours, and this was used as a seed culture. Next, 19 liters of synthetic medium [(NH 4 ) 2 SO 4 7.1 g / l, K 2 HPO 4 1.7 g / l, casamino acid 5 g / l
l, tryptophan 0.5g / l, glucose 20g / l, MgSO 4 · 7
H 2 0 2.1g / l, CaCl 2 0.19g / l, FeCl 3・ 6H 2 O 0.0048g
/ l, thiamine hydrochloride 0.02g / l, H 3 BO 3 4.96 μg / l, (NH 4 ) 6
M 07 O 24 · 4H 2 O 0.74μg / l, CuSO 4 · 5H 2 O 0.5 μg /
l, MnCl 2・ 4H 2 O 4.3 μg / l, ZnSO 4・ 7H 2 0 0.58 μg / l)
1 l of the above seed culture solution was added to the above and cultured at pH 7.0 and 37 ° C. for 24 hours using a 30 l fermenter. At 12 hours, 800 g of glucose powder was added.
【００１５】24時間の培養終了後、培養液を１ｌずつ４
つに分け、硫酸を用いてそれぞれpH3.0, pH 4.0, pH 5.
0,に調整し、さらに他の１つはそのまま(pH 7.0)で室温
で２時間放置した。その後、各培養液の遠心上清を滅菌
水で適当に希釈し、寒天培地(Bacto-Tryptone 10g/l, B
acto-Yeast extract 5g/l, NaCl 10g/l, Bacto-Agar15g
/l)を入れたプレート上にまき、37℃で一晩インキュベ
ートし生成したコロニーを計数して生菌数及びその活性
を求めた。その結果は、次に示すように、pH4.0 で99％
の大腸菌が、pH 3.0では99.9999 ％以上の大腸菌が死滅
した。ヒルジンの活性は特願平2-303096に記載の方法で
測定し、pH 3.0の場合でも90％以上の活性が保持されて
いることを確認した。After culturing for 24 hours, 1 l of the culture solution was added to each well.
PH 3.0, pH 4.0, pH 5.
It was adjusted to 0, and the other one was left as it was (pH 7.0) at room temperature for 2 hours. After that, the supernatant of each culture was appropriately diluted with sterilized water, and agar medium (Bacto-Tryptone 10 g / l, B
acto-Yeast extract 5g / l, NaCl 10g / l, Bacto-Agar 15g
/ l) was placed on the plate and incubated overnight at 37 ° C., and the produced colonies were counted to determine the viable cell count and its activity. The results are 99% at pH 4.0, as shown below.
E. coli, but at pH 3.0, more than 99.9999% of E. coli was killed. The activity of hirudin was measured by the method described in Japanese Patent Application No. 2-303096, and it was confirmed that 90% or more of the activity was retained even at pH 3.0.
【００１６】 pH 死 滅 率 活 性 7.0 0％ 100％ 5.0 90％ 95.6％ 4.0 99％ 96.5％ 3.0 >99.9999 ％ 91.8％PH mortality Activity 7.0 0% 100% 5.0 90% 95.6% 4.0 99% 96.5% 3.0> 99.9999% 91.8%
【００１７】[0017]
【実施例２】実施例１と同様にして調整したpH 3.0, 4.
0, 5.0, 7.0 のヒルジン分泌生産菌の培養液を室温で２
時間放置後、各 500mlを 6000rpmで30分遠心分離し、そ
の上清を40％NaOHでpH 7.0に中和した。これをポアサイ
ズ３μのフィルターでブフナーロートで濾過し、その濾
液をポアサイズ 0.8μのフィルターで濾過し、その濾液
を 0.2μのフィルターで濾過した。各フィルターが目づ
まりし濾過できなくなるまでに濾過した液量を測定し
た。フィルターはアドバンテックトーヨー製の47mmφの
セルロースニトレート(3μ) 及びセルロースアセテート
(0.8μと 0.2μ)を用いた。Example 2 The pH adjusted to 3.0, 4.
Culture solution of 0, 5.0, 7.0 hirudin secretory producers at room temperature
After leaving it for an hour, 500 ml of each was centrifuged at 6000 rpm for 30 minutes, and the supernatant was neutralized to pH 7.0 with 40% NaOH. This was filtered through a Buchner funnel with a filter having a pore size of 3μ, the filtrate was filtered through a filter having a pore size of 0.8μ, and the filtrate was filtered through a filter of 0.2μ. The amount of liquid filtered until each filter became clogged and filtration became impossible was measured. Filter is Advantech Toyo's 47mmφ cellulose nitrate (3μ) and cellulose acetate
(0.8μ and 0.2μ) were used.
【００１８】その結果、次に示すように、pH 4.0及び
3.0で処理したものはその濾過性が向上しフィルターが
目づまりしにくいことがわかった。 pH ３μフィルター(ml) 0.8μフィルター(ml) 0.2μフィルター(ml) 7.0 150 50 10 5.0 150 50 10 4.0 250 150 50 3.0 250 350 350As a result, as shown below, pH 4.0 and
It was found that those treated with 3.0 had improved filterability and were less likely to clog the filter. pH 3μ filter (ml) 0.8μ filter (ml) 0.2μ filter (ml) 7.0 150 50 10 5.0 150 50 10 4.0 250 150 50 3.0 250 350 350
【００１９】[0019]
【実施例３】実施例１と同様にヒルジン分泌生産菌を培
養したところ、ある培養ではその培養後期に培地中のヒ
ルジンの活性が低下し、24時間目に活性がまったく失わ
れてしまった。これは大腸菌のプロテアーゼが何らかの
条件で誘導され、それが培地中のヒルジンを分解したた
めと考えられる。[Example 3] When a hirudin-secreting producing bacterium was cultured in the same manner as in Example 1, the activity of hirudin in the medium decreased in a certain culture in the latter half of the culture, and the activity was completely lost at 24 hours. This is probably because E. coli protease was induced under some conditions, which decomposed hirudin in the medium.
【００２０】この異常培養の培養液上清と正常な培養の
培養液上清を１：１で混合しH2SO4 でpHを 5.0, 4.0 に
調整し、37℃又は４℃で一夜インキュベートしてその活
性を調べてみた。その結果、pH 7.0では99％の活性が失
われ、また４℃では93％活性が残存したが、pH 4.0では
97％以上の活性が残っていた。また、pH 4.0に調整し、
一夜放置後、pH 7.0にもどし37℃で三夜放置した場合で
あっても約70％近く活性が存在していた。これらはpH
4.0にすることによりプロテアーゼが失活したためと考
えられる。The culture supernatant of the abnormal culture and the culture supernatant of the normal culture were mixed at a ratio of 1: 1 and the pH was adjusted to 5.0 and 4.0 with H 2 SO 4 , and the mixture was incubated at 37 ° C or 4 ° C overnight. I checked its activity. As a result, 99% activity was lost at pH 7.0 and 93% activity remained at 4 ° C, but at pH 4.0
Over 97% activity remained. Also, adjust to pH 4.0,
Even after being left overnight, the pH was returned to 7.0 and left at 37 ° C. for three nights. These are pH
It is considered that protease was inactivated by setting to 4.0.
【００２１】 pH インキュベート 活 性 正常培養１／２希釈 7.0 ── 100％ 異常 〃 〃 8.0 ── 0％ 正常と異常を１：１混合 7.0 4℃一夜 93.6％ 〃 7.0 37℃一夜 0.9％ 〃 5.0 37℃一夜 27.5％ 〃 4.0 37℃一夜 97.8％ 〃 をpH7.0 にもどし37℃三夜 69.8％PH Incubation Activity Normal culture 1/2 dilution 7.0 ─ 100% Abnormal 〃 〃 8.0 ─ ─ 0% Normal and abnormal 1: 1 mixture 7.0 4 ° C overnight 93.6% 〃 7.0 37 ° C 0.9% 〃 5.0 37 ℃ overnight 27.5% 〃 4.0 37 ℃ overnight 97.8% 〃 returns to pH 7.0 37 ℃ 3 nights 69.8%
【００２２】[0022]
【実施例４】実施例１と同様に調製した24時間後の培養
液の一部を H2SO4でpH 3.5に調整し、室温で１時間放置
後遠心分離して上清を得た。これと培養終了時の培養液
の遠心上清を８−25％のグラジエントSDS-ポリアクリル
アミド電気泳動を行ない、クマシーブルーで染色後デン
シトメーターで分析を行なった。その結果、培養終了時
の培養上清中ヒルジンのタンパク純度は約30％であるの
に対し、pH 3.5で処理したものは分子量の大きいタンパ
クが除去されヒルジンのタンパク純度は約70％と向上し
ていた。Example 4 A part of the culture solution prepared after 24 hours and adjusted in the same manner as in Example 1 was adjusted to pH 3.5 with H 2 SO 4 , allowed to stand at room temperature for 1 hour and then centrifuged to obtain a supernatant. The centrifugation supernatant of this and the culture solution at the end of the culture was subjected to 8-25% gradient SDS-polyacrylamide electrophoresis, stained with Coomassie blue and analyzed with a densitometer. As a result, the protein purity of hirudin in the culture supernatant at the end of the culture was approximately 30%, whereas those treated with pH 3.5 removed proteins with large molecular weight, and the protein purity of hirudin improved to approximately 70%. Was there.
【００２３】[0023]
【発明の効果】本発明は、培養液中のヒルジン類を菌体
及び不純物タンパクから効率よく分離することができ、
また分離に際してヒルジン類の失活を防止できるという
格別の効果を奏する。INDUSTRIAL APPLICABILITY The present invention can efficiently separate hirudins in a culture solution from bacterial cells and impurity proteins.
In addition, it has a special effect of preventing deactivation of hirudins upon separation.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.5 識別記号 庁内整理番号 ＦＩ 技術表示箇所 (Ｃ１２Ｐ 21/02 Ｃ１２Ｒ 1:19） (72)発明者 古屋 英之 埼玉県戸田市新曽南三丁目17番35号 日本 鉱業株式会社内─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. 5 Identification number Reference number within the agency FI technology display location (C12P 21/02 C12R 1:19) (72) Inventor Hideyuki Furuya, 3rd Niizominami, Toda City, Saitama Prefecture 17th 35th Japan Mining Co., Ltd.
Claims (3)
直後の菌体を含む培養液を酸によりpH５以下に調整し、
暫時放置後、これを菌体と培養上清とに分離することを
特徴とするヒルジン類分泌生産培養液の処理方法。1. A pH value of the culture solution containing the cells immediately after the culture of the hirudin secretory production culture solution is adjusted to 5 or less with an acid,
A method for treating a hirudin secretory production culture solution, which comprises leaving the cells for a while and separating them into a bacterial cell and a culture supernatant.
腸菌である請求項１記載の処理方法。2. The treatment method according to claim 1, wherein the hirudin secretory producing bacterium is genetically modified Escherichia coli.
ことである請求項１または２に記載の処理方法。3. The treatment method according to claim 1, wherein the adjustment of the pH with an acid is to adjust the pH to 3 to 4.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3257012A JP2573114B2 (en) | 1991-09-09 | 1991-09-09 | Treatment of hirudin secretion production culture solution |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3257012A JP2573114B2 (en) | 1991-09-09 | 1991-09-09 | Treatment of hirudin secretion production culture solution |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0568582A true JPH0568582A (en) | 1993-03-23 |
| JP2573114B2 JP2573114B2 (en) | 1997-01-22 |
Family
ID=17300505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3257012A Expired - Fee Related JP2573114B2 (en) | 1991-09-09 | 1991-09-09 | Treatment of hirudin secretion production culture solution |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2573114B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5972648A (en) * | 1993-09-28 | 1999-10-26 | Japan Energy Corporation | Hirudin analogs, methods of manufacture thereof and anticoagulant compositions having these as active ingredients |
| WO2011043443A1 (en) * | 2009-10-07 | 2011-04-14 | 三菱化学株式会社 | Method for producing aliphatic dicarboxylic acid |
| JP2013507213A (en) * | 2009-10-13 | 2013-03-04 | バイエル・ファルマ・アクチェンゲゼルシャフト | Method for inactivating undesirable contaminants in medical leech extract |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61501609A (en) * | 1984-03-27 | 1986-08-07 | トランスジ−ン ソシエテ アノニム | Vector for expression of hirudin, transformed cells and method for producing hirudin |
-
1991
- 1991-09-09 JP JP3257012A patent/JP2573114B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61501609A (en) * | 1984-03-27 | 1986-08-07 | トランスジ−ン ソシエテ アノニム | Vector for expression of hirudin, transformed cells and method for producing hirudin |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5972648A (en) * | 1993-09-28 | 1999-10-26 | Japan Energy Corporation | Hirudin analogs, methods of manufacture thereof and anticoagulant compositions having these as active ingredients |
| WO2011043443A1 (en) * | 2009-10-07 | 2011-04-14 | 三菱化学株式会社 | Method for producing aliphatic dicarboxylic acid |
| JP2013507213A (en) * | 2009-10-13 | 2013-03-04 | バイエル・ファルマ・アクチェンゲゼルシャフト | Method for inactivating undesirable contaminants in medical leech extract |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2573114B2 (en) | 1997-01-22 |
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