JPH03244390A - Method for removing substance derived from escherichia coli - Google Patents

Method for removing substance derived from escherichia coli

Info

Publication number
JPH03244390A
JPH03244390A JP2041143A JP4114390A JPH03244390A JP H03244390 A JPH03244390 A JP H03244390A JP 2041143 A JP2041143 A JP 2041143A JP 4114390 A JP4114390 A JP 4114390A JP H03244390 A JPH03244390 A JP H03244390A
Authority
JP
Japan
Prior art keywords
escherichia coli
aqueous solution
substance derived
derived
resin
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
Application number
JP2041143A
Other languages
Japanese (ja)
Inventor
Junichi Kajiwara
淳一 梶原
Mitsuo Enomoto
榎本 光生
Kenkichi Takagi
健吉 高木
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nippon Kayaku Co Ltd
Original Assignee
Nippon Kayaku Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Nippon Kayaku Co Ltd filed Critical Nippon Kayaku Co Ltd
Priority to JP2041143A priority Critical patent/JPH03244390A/en
Publication of JPH03244390A publication Critical patent/JPH03244390A/en
Pending legal-status Critical Current

Links

Classifications

    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/52Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Landscapes

  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

PURPOSE:To remove a substance derived from Escherichia coli from an aqueous solution of genetic recombinant proteins, etc., by bringing an aqueous solution of the substance derived from the Escherichia coli into contact with a strong basic anion exchange resin and adsorbing the substance derived from the Escherichia coli on the aforementioned resin. CONSTITUTION:An aqueous solution containing both microbial cell proteins derived from Escherichia coli and pyrogens is brought into contact with a strong basic anion exchange resin to adsorb a substance derived from the Escherichia coli on the aforementioned resin and isolate the substance derived from the Escherichia coli. The aqueous solution containing the substance derived from the Escherichia coli is normally an aqueous solution containing proteins produced by genetic recombination using the Escherichia coli as a host. In this case, a supernatant or partially purified aqueous solution after crushing the microbial cells is cited as the aqueous solution. A strong basic anion exchange resin mainly having quaternary amines as exchange groups is preferably used as the strong basic anion exchange resin.

Description

【発明の詳細な説明】 〈産業上の利用分野〉 本発明は例えば遺伝子操作技術により製造した蛋白水溶
液中の宿主(大腸菌)由来の物質の除去などに利用でき
るものである。
DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention can be used, for example, to remove host (E. coli)-derived substances from an aqueous protein solution produced by genetic engineering technology.

く従来の技術〉 近年生体内の生理活性蛋白なとを遺伝子操作の手法を用
いて大量に生産させ、医薬品として使用するケースか急
増しているかこの場合は従来の生体組織等から抽出して
いたのと異なり宿主として用いる微生物由来の成分、特
に菌体蛋白及び発熱性物質か蛋白中に混入してくること
か安全性の面から好ましくないとされている。よってこ
のような菌体由来の物質の混入を極めて微量に抑える必
要性か生じてきたかこれまでにこれらを効率的に除く方
法はなく種々のカラムクロマトグラフィーを組み合わせ
るなど極めて煩雑な方法しか知られてないない。
(Conventional technology) In recent years, there has been a rapid increase in the number of cases in which biologically active proteins are produced in large quantities using genetic engineering techniques and used as medicines. Unlike this, it is considered undesirable from a safety standpoint because components derived from the microorganism used as a host, especially bacterial cell proteins and pyrogens, are mixed into the protein. Therefore, there has been a need to suppress the contamination of such substances derived from bacterial cells to an extremely small amount.To date, there has been no efficient method for removing these substances, and only extremely complicated methods such as combining various column chromatography methods are known. No, no.

〈発明か解決しようとする課題〉 そこで遺伝子組換えの宿主として最も汎用性の高い大腸
菌を用いて生産された遺伝子組換え蛋白などの水溶液か
ら大腸菌由来物質を効率的に除去する方法が求められて
いる。
<Invention or problem to be solved> Therefore, there is a need for a method to efficiently remove E. coli-derived substances from an aqueous solution of genetically modified proteins produced using E. coli, which is the most versatile host for genetic recombination. There is.

〈課題を解決するための手段〉 本発明者らはこれらの問題を解決することを目的として
鋭意研究を行った結果、強塩基性陰イオン交換樹脂に上
記の大腸菌由来物質か選択的に吸着され、極めて効率的
に目的とする蛋白質画分よりこれらを除去できることを
見い出し、本発明を完成した。即ち本発明は、大腸菌由
来物質特に大腸菌由来の菌体蛋白及び発熱性物質(パイ
ロジェン)の両者を含む水溶液を、強塩基性陰イオン交
換樹脂に接触させ、該樹脂にこれらの大腸菌由来物質を
吸着させることを特徴とする大腸菌由来物質除去方法に
関するものである。
<Means for Solving the Problems> As a result of intensive research aimed at solving these problems, the present inventors found that the above-mentioned E. coli-derived substances can be selectively adsorbed to a strongly basic anion exchange resin. have discovered that these can be removed from the target protein fraction extremely efficiently, and have completed the present invention. That is, the present invention involves contacting an aqueous solution containing both Escherichia coli-derived substances, particularly Escherichia coli-derived bacterial cell proteins and pyrogens, with a strongly basic anion exchange resin, and adsorbing these Escherichia coli-derived substances on the resin. The present invention relates to a method for removing Escherichia coli-derived substances, which is characterized by:

本発明で除去する大腸菌由来物質の代表的なものとして
は菌体蛋白及び発熱性物質(パイロシュン)かあげられ
、本発明によればこれらの両物質が同時に除かれる点て
非常に画期的である。
Typical E. coli-derived substances that are removed by the present invention include bacterial protein and pyrogenic substances, and the present invention is extremely innovative in that both of these substances are removed at the same time. be.

本発明で使用する大腸菌由来物質を含む水溶液としては
大腸菌由来物質を不純物として含む水溶液ならば制限は
ないか、通常は大腸菌を宿主として遺伝子組換えにより
生産した蛋白を含む水溶液かあげられる。このような遺
伝子組換蛋白水溶液の場合には、菌体破砕後の上清、部
分精製したものなとか挙げられるか粗精製したものをか
けるのか効率的である。
The aqueous solution containing the E. coli-derived substance used in the present invention is not limited as long as it contains the E. coli-derived substance as an impurity, and usually includes an aqueous solution containing a protein produced by genetic recombination using E. coli as a host. In the case of such an aqueous recombinant protein solution, it is efficient to use the supernatant after disrupting the bacterial cells, partially purified, or roughly purified.

また大腸菌由来物質を含む水溶液か、上記の遺伝子組換
え蛋白水溶液のように有用物質の水溶液の場合には、含
有される有用物質は、大腸菌由来の菌体蛋白及び発熱物
質を強塩基性陰イオン交換樹脂に吸着させる際に、該樹
脂に吸着されにくいものであることか必要である。本発
明者らは検討によると、これらの大腸菌由来物質は、か
なり低いpHても該樹脂に吸着されるので酸性側の条件
でクロマトを行うことか可能である。従って、含有され
る有用物質は弱酸性〜塩基性の物質、例えばヒト型銅−
亜鉛スーパーオキシドディスムターゼやリゾチームなと
の蛋白かあげられる。
In addition, in the case of an aqueous solution containing an E. coli-derived substance or an aqueous solution of a useful substance such as the genetically modified protein aqueous solution mentioned above, the useful substance contained is a strong basic anion that binds the E. coli-derived bacterial protein and pyrogen. When it is adsorbed onto a replacement resin, it must be difficult to be adsorbed by the resin. According to studies conducted by the present inventors, these Escherichia coli-derived substances are adsorbed to the resin even at a fairly low pH, so it is possible to perform chromatography under acidic conditions. Therefore, the useful substances contained are weakly acidic to basic substances, such as human copper.
Examples include proteins such as zinc superoxide dismutase and lysozyme.

本発明で使用する強塩基性陰イオン交換樹脂としては大
腸菌由来物質か吸着するものであれば特に制限はないか
主として4級アミン類を交換基としたもので例えばトリ
エチルアミノエチル基を導入したQAE−)ヨパール5
50C、トリメチルアミノエチル基を導入したQAE−
23W  (東ソ、−株式会社)、トリメチルアミノメ
チル基を導入したMCI GEL CAシリーズ、5A
IIA 、 5A12A 5SAIOA 、 PA30
6 、PA312 、PA316 、PA318 、H
PA25  (三菱化成工業株式会社)、ジエチル−2
−ヒドロキシプロピルアミノエチル基を導入したQAE
セファデックスA−25、A−50、Q−セファロース
(ファルマシア)、ジメチルヒドロキシエチルアミノ基
を導入したセパビーズFP−QA(三菱化成株式会社)
なとかある。
The strongly basic anion exchange resin used in the present invention is not particularly limited as long as it can adsorb E. coli-derived substances, or it may be one that mainly uses quaternary amines as an exchange group, such as QAE with a triethylaminoethyl group introduced. -) Yopal 5
50C, QAE- with trimethylaminoethyl group introduced
23W (Toso, Co., Ltd.), MCI GEL CA series with trimethylaminomethyl group introduced, 5A
IIA, 5A12A 5SAIOA, PA30
6, PA312, PA316, PA318, H
PA25 (Mitsubishi Chemical Industries, Ltd.), diethyl-2
-QAE with hydroxypropylaminoethyl group introduced
Sephadex A-25, A-50, Q-Sepharose (Pharmacia), Sepabeads FP-QA with dimethylhydroxyethylamino group introduced (Mitsubishi Kasei Corporation)
There is such a thing.

本発明の方法は、例えば上記樹脂を、0〜200mM濃
度好ましくはO〜50mM濃度の中性塩(例えば塩化ナ
トリウム、塩化カリウムなと)を含むpH約2〜約9好
ましくはpH約2〜約6の緩衝液で平衡化した後、大腸
菌由来物質を含む水溶液(同一な緩衝液の溶液)を、該
樹脂のカラムに通すと、上記の大腸菌由来物質が該樹脂
に吸着され、除去される。該水溶液か前記の有用物質を
含む場合には、該有用物質は素通りした水溶液中に含ま
れるので、平衝化に用いたのと同一な緩衝液を樹脂量の
3〜5倍流して洗浄し、非吸着の有用物質を回収する。
The method of the invention includes, for example, treating the resin at a pH of about 2 to about 9, preferably at a pH of about 2 to about 9, containing a neutral salt (e.g., sodium chloride, potassium chloride, etc.) at a concentration of 0 to 200 mM, preferably from 0 to 50 mM. After equilibration with the buffer solution in step 6, when an aqueous solution containing the E. coli-derived substance (a solution of the same buffer solution) is passed through the column of the resin, the E. coli-derived substance is adsorbed to the resin and removed. If the aqueous solution contains the above-mentioned useful substance, the useful substance will be included in the aqueous solution that passes through, so wash it by flowing 3 to 5 times the amount of the resin as the same buffer used for equilibration. , to recover non-adsorbed useful substances.

上記緩衝液としては上記pH範囲に調整できるものであ
れば特に制限はなく、酢酸系、クエン酸系、リン酸系な
といずれも使用できる。
The buffer solution is not particularly limited as long as it can be adjusted to the above pH range, and acetic acid, citric acid, and phosphoric acid buffers can be used.

なお、該樹脂に吸着された大腸菌由来の菌体蛋白及び発
熱性物質は、緩衝液の塩濃度を例えば約IM濃度程度に
あげてカラムに通液することにより溶出されるので、核
樹脂は繰返し使用することかてきる。
Note that the bacterial cell proteins and pyrogens derived from E. coli adsorbed on the resin are eluted by increasing the salt concentration of the buffer solution to, for example, about IM concentration and passing the solution through the column. You can use it.

本発明を実施例により更に詳細に説明するか本発明の範
囲はこれらの例に限定されるものではない。
The present invention will be explained in more detail with reference to examples, but the scope of the present invention is not limited to these examples.

(実施例1) QAE−トヨパールの樹脂100−に10mM酢酸緩衝
液(pH4,0)を50(7’流してカラムを平衡化し
た。
(Example 1) A column was equilibrated by flowing 50 (7') of 10 mM acetate buffer (pH 4,0) onto QAE-Toyopearl resin 100-.

大腸菌て生産させたヒト型銅−亜鉛スーパーオキシドデ
ィスムターセ(以下r−hsODと略す)100万単位
を含む粗精製液10(7’を同上の緩衝液に透析した後
カラムにかけ続いて500rIL1の同緩衝液で洗浄し
た。この素通り画分に回収されたr−hsODの活性回
収率は95%であり、比活性は4000U/mg蛋白て
あった。また5DS−電気泳動て精製r−hsODは還
元条件下で16にダルトンの単一ハンドを示した。さら
に表1に示したように大腸菌由来蛋白は本カラム精製前
の0.51O−3(%)、発熱性物質は0.13%に減
少していた。なお本実施例においてSOD活性はMcC
ordとFr1dorichの方法(JBC24460
49(1969))により、蛋白定量はLowryらの
方法(JBC193 265(1951))により行っ
た。大腸菌由来蛋白の測定は大腸菌成分を免疫して得ら
れたポリクローナル抗体を用いた酵素免疫測定法により
、発熱性物質の測定はパイロデイック(生化学工業株式
会社)により行った。
Crude purified solution 10 (7') containing 1 million units of human copper-zinc superoxide dismutase (hereinafter abbreviated as r-hsOD) produced by Escherichia coli was dialyzed against the same buffer solution, applied to a column, and then purified with 500 rIL1. It was washed with the same buffer. The activity recovery rate of r-hsOD recovered in this flow-through fraction was 95%, and the specific activity was 4000 U/mg protein. In addition, purified r-hsOD was purified by 5DS-electrophoresis. Dalton's single hand was shown in 16 under reducing conditions.Furthermore, as shown in Table 1, E. coli-derived protein was 0.51O-3 (%) before purification with this column, and pyrogenic substances were 0.13%. In this example, SOD activity was decreased by McCC.
ord and Fr1dorich method (JBC24460
49 (1969)), and protein quantification was performed by the method of Lowry et al. (JBC193 265 (1951)). E. coli-derived proteins were measured by enzyme immunoassay using polyclonal antibodies obtained by immunization with E. coli components, and pyrogenic substances were measured by Pyrodic (Seikagaku Corporation).

(実施例2) QAEセファデックスA−50の樹脂20−に20mM
酢酸緩衝液(pH5,0)を100mA流してカラムを
平衝化した。ニワトリ卵白より精製されたリゾチーム2
0万単位を含む溶液10m1に大腸菌の破砕抽出液5−
を加えて混合した後同上の緩衝液に透析し、カラムにか
けた。続いて100−の同緩衝液で洗浄したところこの
素通り画分にリゾチームが溶出し、回収率は98%てあ
った。このものは5DS−電気泳動て単一バンドとなり
、大腸菌由来蛋白はカラム精製前の0.I Xl0−3
%に、発熱性物質は0.190に減少していた。なお本
実施例においてリゾチーム活性はShugarの方法(
BBA 8302(1952) )により測定した。池
の蛋白定量等は実施例1に記載した方法により行った。
(Example 2) 20mM in QAE Sephadex A-50 resin 20-
The column was equilibrated by flowing an acetate buffer (pH 5,0) at 100 mA. Lysozyme 2 purified from chicken egg white
To 10 ml of solution containing 100,000 units, add 5-
After adding and mixing, the mixture was dialyzed against the same buffer and applied to a column. Subsequently, when the sample was washed with the same 100-ml buffer, lysozyme was eluted in the flow-through fraction, and the recovery rate was 98%. This product was subjected to 5DS-electrophoresis, resulting in a single band, and the E. coli-derived protein was found to be 0.0% before column purification. I Xl0-3
%, pyrogenic substances had decreased to 0.190. In this example, lysozyme activity was determined by Shugar's method (
BBA 8302 (1952)). Quantification of protein in the pond was carried out by the method described in Example 1.

結果は表2によとめた通りである。The results are shown in Table 2.

〈発明の効果〉 実施例1て用いた組換えCu−ZnhSODは等電点か
4.9〜5.2であり、実施例2て用いたリゾチームは
等電点か11.0であったか共に良好な結果か得られた
。この例からもクロマトのpH条件を調整することによ
り種々の蛋白に対して普遍的に適用できることか示され
た。
<Effect of the invention> The recombinant Cu-ZnhSOD used in Example 1 had an isoelectric point of 4.9 to 5.2, and the isoelectric point of lysozyme used in Example 2 had an isoelectric point of 11.0, both of which were good. I got some good results. This example also shows that the method can be universally applied to various proteins by adjusting the pH conditions of the chromatography.

Claims (1)

【特許請求の範囲】[Claims] 1、大腸菌由来物質を含む水溶液を、強塩基性陰イオン
交換樹脂に接触させ、該樹脂に大腸菌由来物質を吸着さ
せることを特徴とする大腸菌由来物質の除去方法
1. A method for removing Escherichia coli-derived substances, which comprises bringing an aqueous solution containing Escherichia coli-derived substances into contact with a strongly basic anion exchange resin, and adsorbing the Escherichia coli-derived substances onto the resin.
JP2041143A 1990-02-23 1990-02-23 Method for removing substance derived from escherichia coli Pending JPH03244390A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2041143A JPH03244390A (en) 1990-02-23 1990-02-23 Method for removing substance derived from escherichia coli

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2041143A JPH03244390A (en) 1990-02-23 1990-02-23 Method for removing substance derived from escherichia coli

Publications (1)

Publication Number Publication Date
JPH03244390A true JPH03244390A (en) 1991-10-31

Family

ID=12600200

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2041143A Pending JPH03244390A (en) 1990-02-23 1990-02-23 Method for removing substance derived from escherichia coli

Country Status (1)

Country Link
JP (1) JPH03244390A (en)

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