JPH01165394A - Method for renaturing insoluble foreign protein - Google Patents
Method for renaturing insoluble foreign proteinInfo
- Publication number
- JPH01165394A JPH01165394A JP62321352A JP32135287A JPH01165394A JP H01165394 A JPH01165394 A JP H01165394A JP 62321352 A JP62321352 A JP 62321352A JP 32135287 A JP32135287 A JP 32135287A JP H01165394 A JPH01165394 A JP H01165394A
- Authority
- JP
- Japan
- Prior art keywords
- protein
- solution
- denaturing agent
- insoluble
- proteins
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 61
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 60
- 238000000034 method Methods 0.000 title claims abstract description 13
- 230000003381 solubilizing effect Effects 0.000 claims abstract description 5
- 239000003795 chemical substances by application Substances 0.000 abstract description 15
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 abstract description 3
- 238000000502 dialysis Methods 0.000 abstract description 3
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 abstract description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 2
- 239000004202 carbamide Substances 0.000 abstract description 2
- 239000012535 impurity Substances 0.000 abstract description 2
- 150000002357 guanidines Chemical class 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 32
- 239000003398 denaturant Substances 0.000 description 14
- 229960000789 guanidine hydrochloride Drugs 0.000 description 14
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 14
- 230000007928 solubilization Effects 0.000 description 7
- 238000005063 solubilization Methods 0.000 description 7
- 238000004925 denaturation Methods 0.000 description 6
- 230000036425 denaturation Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 102000003990 Urokinase-type plasminogen activator Human genes 0.000 description 5
- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229960005356 urokinase Drugs 0.000 description 4
- 239000012528 membrane Substances 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- -1 sulfhydryl compound Chemical class 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Classifications
-
- Y—GENERAL 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements 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
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は宿主細胞において生産された不溶性異種蛋白質
の復元法に関する。DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for restoring insoluble heterologous proteins produced in host cells.
（従来の技術）
遺伝子組換技術のめざましい進展により、さまざまな異
種蛋白質を微生物等を宿主として生産させることか可能
となり、従来その生産性の困難さや収率の低さ等のため
非常に高価であった生理活性物質等を安価に、Ｌかも多
量に供給することが可能になった。(Conventional technology) With the remarkable progress of genetic recombination technology, it has become possible to produce various heterologous proteins using microorganisms as hosts. It has now become possible to supply physiologically active substances and the like in large quantities at low cost.
宿主細胞において生産された異種蛋白質は顆粒状の塊を
形成するなどしばしば不溶性蛋白質として宿主細胞内に
蓄積される。これらの蛋白質は本来の活性を発現し得な
い形態に折り畳まれていることが多く、そのため、特開
昭５９−１６’１３２１号に示された様な変性可溶化及
び復元（再折畳み）という一連の操作が必要とされる。Heterologous proteins produced in host cells often accumulate in the host cells as insoluble proteins, such as forming granular masses. These proteins are often folded in a form that does not allow them to express their original activity, and therefore a series of denaturation, solubilization and renaturation (refolding) as shown in JP-A-59-16'1321 is required. operation is required.
これは、例えば宿主細胞を破砕した後、遠心分離等の方
法により抽出した不溶性異種蛋白質を高濃度の蛋白質変
性剤溶液に懸濁して変性可溶化し、続いて異種蛋白質含
有溶液中の変性剤濃度を希釈あるいは透析等の方法によ
り低下させるなどして行われる。For example, after disrupting host cells, insoluble heterologous proteins extracted by a method such as centrifugation are suspended in a highly concentrated protein denaturant solution to denature and solubilize, and then the denaturant concentration in the heterologous protein-containing solution is This is done by lowering the amount by dilution or dialysis.
（発明が解決しようとする間層点）
宿主細胞中で異種蛋白質が不溶性異種蛋白質として生産
される時には、蛋白質変性剤を用いた変性可溶化及び復
元のための操作が必要となるが、蛋白質変性剤は、生物
にとって有害な物質であるため廃棄することが出来ない
。本発明者らは、この問題を鋭意検討した結果、不溶性
異種蛋白質を変性可溶化及び復元する操作において使用
する蛋白質変性剤量を極力少なくすることで、これらの
操作により生じる蛋白質変性剤含有溶液量を減少させ、
結果として変性剤を廃棄する操作を減少させる方法を完
成させた。即ち本発明は宿主細胞において生産された不
溶性異種蛋白質を蛋白質変性剤を用いて復元する方法に
おいて、不溶性異種蛋白質の変性可溶化及び復元に用い
た変性剤を異種蛋白質含有溶液から回収し再度用いるこ
とを特徴とする不溶性異種蛋白質の復元法を提供するも
のであり、以下に詳細に説明する。(Interlayer point to be solved by the invention) When a heterologous protein is produced as an insoluble heterologous protein in a host cell, an operation for denaturation solubilization and restoration using a protein denaturing agent is required. These substances cannot be disposed of because they are harmful to living organisms. As a result of intensive investigation into this problem, the present inventors have found that by minimizing the amount of protein denaturant used in the operations of denaturing, solubilizing, and restoring insoluble foreign proteins, the amount of protein denaturant-containing solution generated by these operations can be reduced. decrease,
As a result, we have completed a method that reduces the number of operations to discard the denaturing agent. That is, the present invention provides a method for denaturing an insoluble heterologous protein produced in a host cell using a protein denaturing agent, in which the denaturing agent used for denaturation, solubilization, and denaturation of the insoluble heterologous protein is recovered from a heterologous protein-containing solution and used again. The present invention provides a method for restoring an insoluble heterologous protein, which is described in detail below.
（問題点を解決するための手段）
本発明は、グアニジン＃Ａ（塩酸塩、硫酸塩、チオシア
ン酸塩）、尿素、チオシアン酸塩（ナトリウム塩、カリ
ウム塩）等の蛋白質変性剤を用いて不溶性異種蛋白質を
変性可溶化及び復元する方法に採用することができる。(Means for Solving the Problems) The present invention uses a protein denaturant such as guanidine #A (hydrochloride, sulfate, thiocyanate), urea, thiocyanate (sodium salt, potassium salt) to It can be employed in methods for denaturing, solubilizing and renaturing heterologous proteins.
宿主とする微生物及び遺伝子組換技術を用いて生産しよ
うとする異種蛋白質に格段の制限はない。従来よく使用
される宿主細胞としては大腸菌が、また、大腸菌で生産
される不溶性異種蛋白質としては、例えばヒト成長蛋白
質変性剤の回収は、異種蛋白質含有溶液、即ち、生産さ
れた不溶性異種蛋白質を変性可溶化させた段階の溶液、
あるいは復元した段階の溶液等から行えばよい。There are no particular restrictions on the host microorganism or the heterologous protein to be produced using genetic recombination technology. Conventionally, Escherichia coli is used as a host cell, and as an insoluble heterologous protein produced by Escherichia coli, for example, recovery of a human growth protein denaturing agent is performed using a heterologous protein-containing solution, that is, the produced insoluble heterologous protein is denatured. solution at the solubilized stage,
Alternatively, it may be carried out from the solution at the reconstituted stage.
蛋白質変性剤を回収するためには、遺伝子組換技術を用
いて生産され、可溶化された異種蛋白質と蛋白質変性剤
を分離可能な、例えば、透析等の方法を用いればよいが
、なかでも操作が簡単でしかも迅速に大量の溶液を処理
できる限外濾過膜を用いることが好ましい。In order to recover the protein denaturant, it is possible to use a method such as dialysis that can separate the protein denaturant from the heterologous protein produced using genetic recombination technology and solubilized. It is preferable to use an ultrafiltration membrane that is simple and can rapidly process a large amount of solution.
回収した蛋白質変性剤含有溶液は、変性剤の濃度が必要
とされる濃度以上であればなんら問題なく再度同種ある
いは他の不溶性異種蛋白質の変性可溶化及び復元の操作
に使用することが可能である。回収した蛋白質変性剤含
有溶液の変性剤濃度が必要とされる濃度以下で、例えば
そのままでは比較的高い蛋白質変性剤濃度を必要とする
変性可溶化の操作に使用できない場合等には、蛋白質変
性剤を透過させない様な限外濾過膜等を用いて変性剤を
濃縮する等すればよい。The recovered protein denaturing agent-containing solution can be used again for denaturation, solubilization, and renaturation of the same or other insoluble heterologous proteins without any problems as long as the concentration of the denaturant is higher than the required concentration. . If the concentration of the denaturant in the recovered protein denaturant-containing solution is below the required concentration, for example, if it cannot be used as it is for denaturation and solubilization operations that require a relatively high concentration of protein denaturant, use the protein denaturant. The denaturing agent may be concentrated using an ultrafiltration membrane or the like that does not allow it to pass through.
回収された蛋白質変性剤含有溶液に低分子の宿主細胞に
由来する蛋白質、あるいは、異種蛋白質に由来するベグ
チド等が含有されている場合にはこれらの夾雑物と蛋白
質変性剤を限外膜等を用いて分離しておくとよい。その
理由としては、復元の操作の時の異種蛋白質溶液中の＠
Ｇ蛋白質■■＠■の濃度が低い程、異種蛋白質の復元率
（生理活性を発現し得る本来の形態に復元（再折畳み）
された異種蛋白質量／溶液中の全異種蛋白質＃）が、高
いこと等が上げられる。If the recovered protein denaturing agent-containing solution contains low-molecular host cell-derived proteins or vegutide derived from foreign proteins, these impurities and the protein denaturing agent are removed using an ultramembrane, etc. It is best to use it to separate it. The reason for this is that @
The lower the concentration of G protein
For example, the amount of foreign protein obtained/total foreign protein in the solution (#) is high.
（発明の効果）
本発明により、不溶性異種蛋白質を変性可溶化及び復元
する操作において使用される蛋白質変性剤を繰返して使
用できる。このことにより、従来廃棄することが出来ず
、焼却することで処分していた蛋白質変性剤の使用量を
減少させ、さらにはそのような蛋白質変性剤の焼却とい
う煩わしい操作を減少させることが可能である。(Effects of the Invention) According to the present invention, a protein denaturing agent used in operations for denaturing, solubilizing, and restoring insoluble heterologous proteins can be used repeatedly. This makes it possible to reduce the amount of protein denaturants used, which could not previously be disposed of and had to be disposed of by incineration, and also to reduce the troublesome operation of incinerating such protein denaturants. be.
以下更に本発明の詳細な説明するために実施例を示すが
、本発明はこれら実施例に限定されるものではない。Examples will be shown below to further explain the present invention in detail, but the present invention is not limited to these Examples.
（実施例）
実施例１
ブーウロキナーゼをコードする遺伝子を導入したプラス
ミド（このプラスミドは工業技術院微生物工業技術研究
所に寄託されており寄託番号８５４１号を付与されてい
る）で形質転換された大腸菌を培養してグーウロキナー
ゼを生産させた。(Example) Example 1 Escherichia coli transformed with a plasmid into which a gene encoding boourokinase was introduced (this plasmid has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology and has been assigned deposit number 8541) was cultured to produce gourokinase.
生産されたブーウロキナーゼは、大腸菌を破砕しＭ塩酸
グアニジン溶液２ｔを添加してブーウロキナーゼを変性
可溶化し、さらに、スルフヒドリル化合物を含む復元液
２４．５１及び８Ｍ塩酸グアニジン溶液五５ｔを添加し
て塩酸グアニジン濃度を１Ｍに低下させて復元操作を行
った。この溶液のウロキナーゼ活性は、１２４０工Ｕ／
−であった。The produced bouurokinase was obtained by crushing Escherichia coli and adding 2 tons of M guanidine hydrochloride solution to denature and solubilize the bouurokinase, and then adding 24.51 tons of renaturing solution containing a sulfhydryl compound and 55 tons of 8M guanidine hydrochloride solution. A restoring operation was performed by lowering the guanidine hydrochloride concentration to 1M. The urokinase activity of this solution is 1240 units/U/
-It was.
続いて分画分子量３万の限外ｐ過膜ｔＴＩＦ−５０ｒｓ
（東ソー■社製）を用いて復元操作後のウロキナーゼ溶
液から１Ｍ塩酸グアニジン溶液３０ｔ−ゼ国際標準品を
対照に合成基質Ｓ−２４４４（ｘａｂｉ社製）を用いて
Ｔ−Ｋｏｈｎｏらの方法を参考に測定した。（Ｂｉｏｔ
ｅｃｈｎｏｌｏｇｙ ６２ａ巻１９８４年）また、この
プ巾ウロキナーゼのプラスミン処理前の分子量は、およ
そ４万７千である。Next, ultrap membrane tTIF-50rs with a molecular weight cutoff of 30,000
Using synthetic substrate S-2444 (manufactured by Xabi Corporation) as a reference, the method of T-Kohno et al. was measured. (Biot
technology, Vol. 62a, 1984) Furthermore, the molecular weight of this broad urokinase before plasmin treatment is approximately 47,000.
実施例２
実施例１で得られた塩酸グアニジン溶液を用いてプｉウ
ロキナーゼの変性可溶化及び復元操作を行った。実施例
１−と同様に７して得られた不溶性画分に実施例１で得
られた塩酸グアニジン溶液ｚ３を及び新たに８Ｍ塩酸グ
アニジン溶液を１．７を添加してブーウロキナーゼを変
性可溶化し、さらに１スルフヒドリル化合物を含む復元
液１２を及び実施例１で得られた塩酸グアニジン溶液１
６ｔを添加して復元操作を行った。この溶液のウロキナ
ーゼ活性は、１３２０工１１Ｆ／−であった。ただし、
復元溶液として、実施例１で用いた復元溶液の２倍の濃
度のものを使用した。Example 2 Using the guanidine hydrochloride solution obtained in Example 1, denatured solubilization and restoration operations of p-i urokinase were performed. The guanidine hydrochloride solution z3 obtained in Example 1 and a new 8M guanidine hydrochloride solution 1.7 were added to the insoluble fraction obtained in step 7 in the same manner as in Example 1- to denature and solubilize boourokinase. Furthermore, a restoring solution 12 containing a sulfhydryl compound and a guanidine hydrochloride solution 1 obtained in Example 1 were added.
A restoration operation was performed by adding 6t. The urokinase activity of this solution was 1320<11F/-. however,
The restoring solution used had twice the concentration of the restoring solution used in Example 1.
実施例５
実施例１と同様にブーウロキナーゼの変性可溶化及び復
元操作を行い、その復元溶液から１Ｍ塩酸グアニジン溶
液３０ｔを得た。この１Ｍ塩酸グアニジン溶液５０ｔを
アスピレータ−を用いて減圧しつつ、９０℃で気化蒸発
させて８Ｍ塩酸グアニジン溶液五７５ｔを得た。この回
収された８Ｍ塩酸グアニジン溶液と新たな８Ｍ塩酸グア
ニジン溶液約１．８ｔを用いて実施例１と同様の操作を
行った。新たな塩酸グアニジン溶液を用いて変性可溶化
及び復元を行った場合のプ斡ウロキナーゼ活性が１３２
０工Ｕ／−であったのに対して回収した塩酸グアニジン
溶液を用いてこれらの操作を行った場合の＠＠ラウロナ
ーゼ活性は１１８０工Ｕ／−であった。Example 5 In the same manner as in Example 1, bouurokinase was denatured and solubilized and reconstituted, and 30 tons of 1M guanidine hydrochloride solution was obtained from the reconstituted solution. 50 tons of this 1M guanidine hydrochloride solution was vaporized at 90° C. while reducing the pressure using an aspirator to obtain 575 tons of an 8M guanidine hydrochloride solution. The same operation as in Example 1 was performed using the recovered 8M guanidine hydrochloride solution and about 1.8 t of fresh 8M guanidine hydrochloride solution. When denatured solubilization and renaturation were performed using fresh guanidine hydrochloride solution, the protein urokinase activity was 132
The lauronase activity was 1180 engineering U/- when these operations were performed using the recovered guanidine hydrochloride solution, whereas it was 0 engineering U/-.
Claims (1)
蛋白質変性剤を用いて復元する方法において、不溶性異
種蛋白質の変性可溶化及び復元に用いた変性剤を異種蛋
白質含有溶液から回収し再度用いることを特徴とする不
溶性異種蛋白質の復元法。(1) In a method for restoring an insoluble heterologous protein produced in a host cell using a protein denaturing agent, the denaturing agent used for denaturing, solubilizing, and restoring the insoluble heterologous protein is recovered from the heterologous protein-containing solution and used again. Characterized method for restoring insoluble heterologous proteins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62321352A JPH01165394A (en) | 1987-12-21 | 1987-12-21 | Method for renaturing insoluble foreign protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP62321352A JPH01165394A (en) | 1987-12-21 | 1987-12-21 | Method for renaturing insoluble foreign protein |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01165394A true JPH01165394A (en) | 1989-06-29 |
Family
ID=18131622
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62321352A Pending JPH01165394A (en) | 1987-12-21 | 1987-12-21 | Method for renaturing insoluble foreign protein |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01165394A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007115682A (en) * | 2005-10-17 | 2007-05-10 | Samsung Electro-Mechanics Co Ltd | Electronic equipment having fuel cell |
-
1987
- 1987-12-21 JP JP62321352A patent/JPH01165394A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007115682A (en) * | 2005-10-17 | 2007-05-10 | Samsung Electro-Mechanics Co Ltd | Electronic equipment having fuel cell |
| US8043756B2 (en) | 2005-10-17 | 2011-10-25 | Samsung Electro-Mechanics Co., Ltd. | Electronic apparatus having fuel cell |
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