JPS6391080A - Purification of human tissue plasminogen activator - Google Patents
Purification of human tissue plasminogen activatorInfo
- Publication number
- JPS6391080A JPS6391080A JP61236720A JP23672086A JPS6391080A JP S6391080 A JPS6391080 A JP S6391080A JP 61236720 A JP61236720 A JP 61236720A JP 23672086 A JP23672086 A JP 23672086A JP S6391080 A JPS6391080 A JP S6391080A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- tpa
- plasminogen activator
- human tissue
- tissue plasminogen
- 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
- 102000003978 Tissue Plasminogen Activator Human genes 0.000 title claims abstract description 11
- 108090000373 Tissue Plasminogen Activator Proteins 0.000 title claims abstract description 11
- 229960000187 tissue plasminogen activator Drugs 0.000 title claims abstract description 11
- 238000000746 purification Methods 0.000 title claims description 21
- 238000001179 sorption measurement Methods 0.000 claims abstract description 9
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 claims abstract description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 8
- JJMDCOVWQOJGCB-UHFFFAOYSA-N 5-aminopentanoic acid Chemical compound [NH3+]CCCCC([O-])=O JJMDCOVWQOJGCB-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960002684 aminocaproic acid Drugs 0.000 claims abstract description 5
- 239000005909 Kieselgur Substances 0.000 claims abstract description 4
- 239000005289 controlled pore glass Substances 0.000 claims abstract description 4
- 239000000741 silica gel Substances 0.000 claims abstract description 4
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 4
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 claims abstract description 3
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims abstract 4
- XDOLZJYETYVRKV-UHFFFAOYSA-N 7-Aminoheptanoic acid Chemical compound NCCCCCCC(O)=O XDOLZJYETYVRKV-UHFFFAOYSA-N 0.000 claims abstract 2
- UQXNEWQGGVUVQA-UHFFFAOYSA-N 8-aminooctanoic acid Chemical compound NCCCCCCCC(O)=O UQXNEWQGGVUVQA-UHFFFAOYSA-N 0.000 claims abstract 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 20
- 239000002253 acid Substances 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 2
- 239000013604 expression vector Substances 0.000 claims description 2
- GYDJEQRTZSCIOI-LJGSYFOKSA-N tranexamic acid Chemical compound NC[C@H]1CC[C@H](C(O)=O)CC1 GYDJEQRTZSCIOI-LJGSYFOKSA-N 0.000 abstract 1
- 229960000401 tranexamic acid Drugs 0.000 abstract 1
- 239000000872 buffer Substances 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 229920002684 Sepharose Polymers 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000004094 surface-active agent Substances 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- 108010039627 Aprotinin Proteins 0.000 description 4
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 229960004405 aprotinin Drugs 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 4
- 235000018977 lysine Nutrition 0.000 description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 4
- 229920000053 polysorbate 80 Polymers 0.000 description 4
- 235000018102 proteins Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- HBOMLICNUCNMMY-KJFJCRTCSA-N 1-[(4s,5s)-4-azido-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1C1O[C@H](CO)[C@@H](N=[N+]=[N-])C1 HBOMLICNUCNMMY-KJFJCRTCSA-N 0.000 description 3
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 239000011701 zinc Substances 0.000 description 3
- 229910052725 zinc Inorganic materials 0.000 description 3
- 239000012531 culture fluid Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- AKVBCGQVQXPRLD-UHFFFAOYSA-N 2-aminooctanoic acid Chemical compound CCCCCCC(N)C(O)=O AKVBCGQVQXPRLD-UHFFFAOYSA-N 0.000 description 1
- 102000013566 Plasminogen Human genes 0.000 description 1
- 108010051456 Plasminogen Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000003287 bathing Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000011210 chromatographic step Methods 0.000 description 1
- 230000001112 coagulating effect Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 101150008740 cpg-1 gene Proteins 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000003760 tallow Substances 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- -1 thiocyanate ions Chemical class 0.000 description 1
- 230000001131 transforming effect Effects 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
- Enzymes And Modification Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、ヒト組織プラスミノゲン活性化因子（以下、
ＴＰＡという）の精製法に係る。更に詳しくは、本発明
は、ＴＰＡを吸着担体に吸着、洗浄後、ω−アミノ酸を
含む溶液で溶出させるＴＰＡの新規精製法に係る。Detailed Description of the Invention (Industrial Application Field) The present invention relates to human tissue plasminogen activator (hereinafter referred to as
(referred to as TPA). More specifically, the present invention relates to a novel method for purifying TPA in which TPA is adsorbed onto an adsorption carrier, washed, and then eluted with a solution containing ω-amino acids.
丁ＰＡは血液中に存在するプラスミノゲンをプラスミン
に変換する作用を有し、種々の血栓症の治療薬として用
いられている。Ding-PA has the effect of converting plasminogen present in the blood into plasmin, and is used as a therapeutic agent for various thrombosis.
（従来の技術〕
ＴＰＡの精製法として、本因子を産生ずる細胞を培養し
、培養液から亜鉛キレート−アガロース、コンカナバリ
ンＡ−アガロース、ゲル濾過によって精製した例が知ら
れているＣ Ｄ、 ０− Ｒｉｊｋｅｎ とり、 Ｃ
ｏ１１ｅｎ （１９８１年）ジャーナル・オブ・バイオ
ロジカル・ケミストリー（Ｊ、 Ｂｉｏｌ、 Ｏｈｅｍ
、 ） 。(Prior art) As a known method for purifying TPA, there is a known example in which cells producing this factor are cultured and purified from the culture solution by gel filtration using zinc chelate-agarose, concanavalin A-agarose, and gel filtration. Rijken Tori, C
o11en (1981) Journal of Biological Chemistry (J, Biol, Ohem
, ).
２５６巻、７０３５頁〕。また’Ｉ’ＰＡはりジン−セ
ファロース（Ｒ，ＰａｄｃｌｉｆｆｅとＴ、 Ｈｅ１ｎ
ｚｅ（１９７８年）アルヒーフス・オブ・バイオケミス
トリー・アンド・バイオフィジックス（Ａｒｃｈ。Volume 256, page 7035]. Also 'I'PA haridine-sepharose (R, Padcliffe and T, He1n
ze (1978) Archifus of Biochemistry and Biophysics (Arch.
Ｅｉｏｃｈｅｍ、 Ｂｉｏｐｈｙｓ、 ） 、 １８９
巻、１８５頁〕、プロテアーゼインヒビター（ｃ、 Ｈ
ｅｕＳＳｅｎら（１９８４年）ザ・ジャーナル・オブ・
バイオロジカル・ケミストリー（Ｊ、 Ｂｉｏｌ、 Ｃ
ｈｅｍ、 ）、 ２５９巻、１１６８５頁〕或いはモノ
クローナル抗体によるアフィニティークロマトグラフィ
ー等が知られている。Eiochem, Biophys, ), 189
Vol., p. 185], protease inhibitors (c, H
euSSen et al. (1984) The Journal of
Biological Chemistry (J, Biol, C
Hem, ), vol. 259, p. 11685] or affinity chromatography using monoclonal antibodies.
（発明が解決しようとする問題点）
ＴＰＡの産生に用いる細胞は、一般にＴＰＡの誘導が無
血清培地で引き起こされる為に、細胞の培養に用いる培
地は主に無血清培地が用いられてきた（ Ｄ、 Ｏ，Ｒ
ｉｊｋｅｎとり、 Ｃｏ１１ｅｎ （１９８１年）ジャ
ーナル・オブ・バイオロジカル・ケミストリー （Ｊ、
Ｂｉｏｌ、 Ｃｈｅｍ、 ）、 ２５６巻、７０８５
頁〕。(Problems to be Solved by the Invention) Since the induction of TPA in cells used for TPA production is generally caused in a serum-free medium, a serum-free medium has been mainly used for cell culture ( D, O, R
Ijken, Co11en (1981) Journal of Biological Chemistry (J,
Biol, Chem, ), vol. 256, 7085
page〕.
無血清培地を用いた場合はＴＰＡは亜鉛キレート−アガ
ロース、コンカナバリンＡ−アガロース、ゲル濾過とい
うクロマトグラフィーのステップで精製される（同上）
。近年、遺伝子組換えの手法を用いて動物培養細胞にＴ
ＰＡを産生させることが可能になった（ Ｒ，Ｊ、 Ｋ
ａｕｆｍａｎら（１９８５年）モレキュラー・アンド場
セルラー囃バイオロジー（Ｍｏ１． Ｃｅ１１． Ｂｉ
ｏｌ、 ） 、 ５巻、１７５０頁〕。遺伝子組換えの
手法によってＴＰＡを産生ずるように形質転換された細
胞は無血清培地ばかりでなく、血清を含む培地において
もＴＰＡを産生ずる。血清を含む培養液からＴＰＡを上
記方法により精製を行っても、血清由来の蛋白が不純蛋
白として精製ＴＰＡ標品に混入する。またＲｉｊｋｅｎ
らの精製法では精製中’Ｉ’ＰＡの凝集沈殿或いはクロ
マトグラフィー担体への吸着を防ぐ為に、用いる緩衝液
に界面活性剤であるツイーン８０を加えているが、ツイ
ーン８０を精製標品から完全に除去することは困難なこ
とである。If a serum-free medium is used, TPA is purified by chromatographic steps of zinc chelate-agarose, concanavalin A-agarose, and gel filtration (ibid.).
. In recent years, genetic recombination techniques have been used to introduce T into cultured animal cells.
It became possible to produce PA (R, J, K
aufman et al. (1985) Molecular and Field Cellular Biology (Mo1. Ce11. Bi
ol, ), vol. 5, p. 1750]. Cells transformed to produce TPA by genetic recombination techniques produce TPA not only in serum-free medium but also in serum-containing medium. Even if TPA is purified from a serum-containing culture solution by the above method, proteins derived from the serum will be mixed into the purified TPA preparation as impure proteins. Also Rijken
In their purification method, Tween 80, a surfactant, is added to the buffer used to prevent 'I'PA from coagulating and precipitating or adsorbing to the chromatography carrier during purification, but Tween 80 is not added to the purified sample. It is difficult to completely remove it.
本発明者らは以上の様な問題点に対処すべく研究を行っ
た結果、無血清培養液ばかりでなく血清を含む培養液か
らでも’Ｉ’ＰＡを高収率に、更に界面活性剤を用いな
い精製法を確立し、本発明に至った。また本発明はＴＰ
Ａの精製標品から界面活性剤の除去法としても利用可能
である。The present inventors conducted research to address the above-mentioned problems and found that 'I'PA can be produced in high yield not only from serum-free culture fluids but also from serum-containing culture fluids, and also using surfactants. We have established a purification method that does not use this method, leading to the present invention. Moreover, the present invention
It can also be used as a method for removing surfactants from purified specimens of A.
（問題点を解決するための手段）
ＴＰＡ生産細胞を含血清培地で培養し、その培養液に含
まれるＴＰＡがクロマトグラフィー担体に吸着され、ま
た溶出されるかを種々のクロマトグラフィー担体につい
て調べた結果、０ＰＧ（コンドロールド・ポアー・グラ
ス）、シリカゲル或いは硅藻土等の硅酸を含む担体に極
めて効果的に吸着され、またω−アミノ酸を含む溶液に
よう高収率で浴出回収されることが見い出された。(Means for solving the problem) TPA-producing cells were cultured in a serum-containing medium, and various chromatography carriers were examined to determine whether TPA contained in the culture solution was adsorbed to and eluted from the chromatography carrier. As a result, it can be adsorbed very effectively on carriers containing silicic acid such as 0PG (chondral pore glass), silica gel, or diatomaceous earth, and can be recovered in a high yield by bathing in a solution containing ω-amino acids. was discovered.
ＴＰＡが吸着され、ω−アミノ酸を含む緩衝液で溶出さ
れる担体としてリジン−セファロースが知られているが
、リジン−セファロースはＴＰＡのリジン結合部位と親
和性を示し、吸着されたＴＰＡは０．０１％ツイーン８
０存在下１０〜２０ｍＭのリジンを含む緩衝液で溶出さ
ｎる。これに対しＣＰＧ等に吸着された’Ｉ’ＦＡは、
０．０１％ツイーン８０存在下５０ ｍＭのリジンを含
む緩衝液でも溶出されない。従って、リジン−セファロ
ースとＣＰＧへのＴＰＡの吸着は、その吸着様式が異な
っていると推定される。Lysine-Sepharose is known as a carrier on which TPA is adsorbed and eluted with a buffer containing ω-amino acids, but lysine-Sepharose exhibits affinity for the lysine binding site of TPA, and the adsorbed TPA has an affinity of 0. 01% Tween 8
Elute with a buffer containing 10-20mM lysine in the presence of 0n. On the other hand, 'I'FA adsorbed to CPG etc.
It is not eluted even in a buffer containing 50 mM lysine in the presence of 0.01% Tween 80. Therefore, it is presumed that the adsorption modes of TPA to lysine-Sepharose and CPG are different.
ＴＰＡの吸着担体としては、ＣＰＧ１シリカゲル或いは
硅藻土が有効であるが、本精製法に使用できる吸着担体
としてはＣＰＧと同様な吸着様式を示す担体ならどのよ
うな担体でも良い。すなわち吸着したＴＰＡが０．０１
％ツイーン８０存在下５０ ｍＭのリジンを含む緩衝液
では溶出されず、１Ｍの６−アミノヘキサン酸を含む緩
衝液或いは塩を含む緩衝液で出出可能な担体であれば本
発明に包含される。CPG1 silica gel or diatomaceous earth is effective as an adsorption carrier for TPA, but any carrier that can be used in this purification method may be used as long as it exhibits the same adsorption mode as CPG. In other words, the amount of adsorbed TPA is 0.01
A carrier that is not eluted with a buffer containing 50 mM lysine in the presence of % Tween 80 and can be eluted with a buffer containing 1 M 6-aminohexanoic acid or a buffer containing a salt is included in the present invention. .
吸着されたＴＰＡはω−アミノ酸を含む溶液で溶出され
るが、ω−アミノ酸としてはＬ−リジン、４−アミツブ
クン酸、５−アミノペンタン酸、６−アミノヘキサン酸
、７−アミツブクン酸、８−アミノオクタン酸或いはト
ランキサム酸が使用できる。The adsorbed TPA is eluted with a solution containing ω-amino acids, and the ω-amino acids include L-lysine, 4-amitubuconic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, 7-amitubucunic acid, and 8-amino acid. Aminooctanoic acid or tranxamic acid can be used.
ＣＰＧ等に吸着されたＴＰＡはＣＰＧとＴＰＡ。TPA adsorbed to CPG etc. is CPG and TPA.
ＴＰＡ間或いはＴＰＡと夾雑蛋白との間の疎水性結合が
存在している可能性があり、チオシアン酸イオンのよう
なカオトロピックなイオンの存在下ではω−アミノ酸を
含む溶液によるＴＰＡの溶出が容易になる。Hydrophobic bonds may exist between TPA or between TPA and contaminant proteins, and in the presence of chaotropic ions such as thiocyanate ions, TPA can be easily eluted with a solution containing ω-amino acids. Become.
本精製法は、界面活性剤の非存在下で実施可能であり、
また界面活性剤を含むＴＰＡ標品から界面活性剤を除去
する方法としても応用が可能である。更に本精製法は、
細胞を培養した培養液を透析等の前処理をすることなく
精製の出発材料として用いることができ、ＴＰＡ精製の
第一段階として非常に有効である。This purification method can be carried out in the absence of surfactants,
It can also be applied as a method for removing surfactants from TPA specimens containing surfactants. Furthermore, this purification method
The culture solution in which cells are cultured can be used as a starting material for purification without pretreatment such as dialysis, and is very effective as the first step in TPA purification.
（実施例） 以下に実施例を示す。(Example) Examples are shown below.
実施例１
ＴＰＡ生産細胞の培養
ＴＰＡ生産細胞ＣＨＯＥＢ４８９９は、特願昭６１−９
７４８１に記載の方法により、ＣＨＯｄｈｆｒ″′株を
’１’ＦＡ発現ベクターｐｓＶｅＰＡ−８により形質転
換して得た細胞である。このＣＨＯＥＢ４８９９株を２
．５％牛脂児血清、２５ＫＩＵ／ｘｔｌアプロチニンを
含むＭＥＭアルファ培地（ＧＩＢＯＯａ−製）テ培養し
、約３０ＯＬ＝ツ）−／ｌｌのＴＰＡ活性を有する培養
液２０ｇを得た。Example 1 Culture of TPA-producing cells TPA-producing cells CHOEB4899 were obtained by patent application 1986-9.
These cells are obtained by transforming the CHOdhfr'' strain with the '1'FA expression vector psVePA-8 by the method described in 7481.
．． The mixture was cultured in MEM alpha medium (manufactured by GIBOOa) containing 5% tallow serum and 25 KIU/xtl aprotinin to obtain 20 g of a culture solution having a TPA activity of approximately 30 OL/l.
実施例２
ＴＰＡの種々のクロマトグラフィー担体による精製
種々のクロマトグラフィー担体をカラム（０，７５×４
備）に詰め、１０ ｍＭリン酸緩衝液（ｐＨ７，５）で
平衡化した後、実施例１で得たＴＰＡを含む培養液を夫
々２０Ｍ１チヤージした。次にＩ Ｍ Ｎａ１ｌ、２
５ ＫＩＵ／ｇ／アプロチニンを含む１０ ｍＭリン酸
緩衝液８ｚｔで洗浄後、更に表１に示した成分を加えた
緩衝液にて溶出した。表１に示したようにＴＰＡはω−
アミノ酸を含む緩衝液で極めて高収率に回収さｎた。精
製培率は約２０培であった。Example 2 Purification of TPA with various chromatography carriers Various chromatography carriers were purified by column (0,75
After equilibration with 10 mM phosphate buffer (pH 7,5), 20 M of each culture solution containing TPA obtained in Example 1 was charged. Then I M Na1l, 2
After washing with 10 mM phosphate buffer 8zt containing 5 KIU/g/aprotinin, elution was further performed with a buffer containing the components shown in Table 1. As shown in Table 1, TPA is ω-
It was recovered in extremely high yield with a buffer containing amino acids. The purification culture ratio was approximately 20 cultures.
表１
実施例３
ＴＰＡのＯＰＧカラムクロマトグラフィーによる精製
ＣＰ Ｇ （ｃｏｎｔｒｏｌｅｄ ｐｏｒｅ ｇｌａｓ
ｓ ： エレクトロヌクレオニクス社）をカラムに詰め
＜２．５Ｘ８０ａｎ）、１０ｍＭリン酸緩衝液（ｐＨ７
，５）で平衡化後、実施例１で得たＴＰＡを含む培養液
６．８４をチャージした。ＩＭ Ｎａ１ｌ、２５ＫＩ
Ｕ／ｒｌアプロチニンを含む２０ ｍＭリン酸緩衝液（
ｐＨ７，５）、次に０．５Ｍ ＫＳＯＮ、ＩＭ Ｎ
ａＣ１゜０．２Ｍ６−アミノヘキサン酸（６−ＡＨ）、
２５ＫＩＵ／ｓ＋／アプロチニンを含む２０ｍＭリン酸
緩衝液（ｐＨ７，５）で順次洗浄後、０．２Ｍから１．
２Ｍの６−アミノヘキサン酸の直線濃度勾配で溶出した
。Table 1 Example 3 Purification of TPA by OPG column chromatography CP G (controlled pore glass)
s: Electronic Nucleonics) packed in a column <2.5X80an), 10mM phosphate buffer (pH 7
, 5), 6.84 g of the TPA-containing culture solution obtained in Example 1 was charged. IM Na1l, 25KI
20 mM phosphate buffer containing U/rl aprotinin (
pH 7,5), then 0.5M KSON, IM N
aC1゜0.2M6-aminohexanoic acid (6-AH),
After sequential washing with 20mM phosphate buffer (pH 7,5) containing 25KIU/s+/aprotinin, 0.2M to 1.
Elution was performed with a linear gradient of 2M 6-aminohexanoic acid.
第１図に示したように、カラムから溶出され、精製倍率
３０倍、回収率９５％で精製された。As shown in FIG. 1, it was eluted from the column and purified with a purification factor of 30 times and a recovery rate of 95%.
実施例４
部分精製ＴＰＡのＣＰＧカラムクロマトグラフィーによ
る精製
実施例１で得た培養液から、常法に従い、ＴＰＡヲ亜鉛
キレート−セファロース、コンカナバリンＡ−セファロ
ース、リジン−セファロースのクロマトグラフィーによ
り部分精製した。ＣＰＣ，をカラムに詰め（２，５Ｘ１
０（７））、部分精製Ｔ Ｐ Ａを１２万ユニツトチヤ
ージした。次に実施例２と同様に洗浄後、ＴＰＡを６−
アミノヘキサン酸（６−ＡＨ）の直線濃度勾配で溶出し
た。第２図に示したように、ＴＰＡは主蛋白ピークとし
て溶出され、精製倍率２．３倍、回収率９３％で精製さ
れた。Example 4 Partial Purification Purification of TPA by CPG Column Chromatography From the culture solution obtained in Example 1, TPA was partially purified by chromatography on zinc chelate-Sepharose, concanavalin A-Sepharose, and lysine-Sepharose according to a conventional method. CPC, packed in a column (2,5X1
0(7)), 120,000 units of partially purified TPA were charged. Next, after washing in the same manner as in Example 2, 6-
Elution was performed with a linear concentration gradient of aminohexanoic acid (6-AH). As shown in FIG. 2, TPA was eluted as the main protein peak and purified with a purification factor of 2.3 times and a recovery rate of 93%.
第１図及び第２図は、ともにＴＰＡのＯＰＧカラムクロ
マトグラフィーの溶出パターンを示す図である。FIG. 1 and FIG. 2 both show the elution pattern of TPA in OPG column chromatography.
Claims (6)
吸着させ、吸着後ω−アミノ酸を含む溶液で溶出するこ
とを特徴とするヒト組織プラスミノゲン活性化因子の精
製法。(1) A method for purifying human tissue plasminogen activator, which comprises adsorbing human tissue plasminogen activator onto an adsorption carrier, and eluting it with a solution containing ω-amino acids after adsorption.
ＣＰＧ）である特許請求の範囲第１項記載の精製法。(2) The adsorption carrier is controlled pore glass (
CPG), the purification method according to claim 1.
請求の範囲第１項記載の精製法。(3) The purification method according to claim 1, wherein the adsorption carrier is silica gel or diatomaceous earth.
酸、５−アミノペンタン酸、６−アミノヘキサン酸、７
−アミノヘプタン酸、８−アミノオクタン酸或いはトラ
ンキサム酸の何れかである特許請求の範囲第１項乃至第
３項の何れかの項記載の精製法。(4) ω-amino acid is L-lysine, 4-aminobutanoic acid, 5-aminopentanoic acid, 6-aminohexanoic acid, 7
-aminoheptanoic acid, 8-aminooctanoic acid or tranxamic acid, the purification method according to any one of claims 1 to 3.
プラスミノゲン活性化因子発現ベクターによって形質転
換された細胞によって産生されたヒト組織プラスミノゲ
ン活性化因子である特許請求の範囲第１項乃至第４項の
何れかの項記載の精製法。(5) Any of claims 1 to 4, wherein the human tissue plasminogen activator is a human tissue plasminogen activator produced by a cell transformed with a human tissue plasminogen activator expression vector. Purification method described in the above section.
養液中に含まれているヒト組織プラスミノゲン活性化因
子である特許請求の範囲第１項乃至第５項の何れかの項
記載の精製法。(6) The purification method according to any one of claims 1 to 5, wherein the human tissue plasminogen activator is a human tissue plasminogen activator contained in a cell culture solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61236720A JPS6391080A (en) | 1986-10-03 | 1986-10-03 | Purification of human tissue plasminogen activator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP61236720A JPS6391080A (en) | 1986-10-03 | 1986-10-03 | Purification of human tissue plasminogen activator |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS6391080A true JPS6391080A (en) | 1988-04-21 |
Family
ID=17004777
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP61236720A Pending JPS6391080A (en) | 1986-10-03 | 1986-10-03 | Purification of human tissue plasminogen activator |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS6391080A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8563288B2 (en) | 2001-05-21 | 2013-10-22 | Omrix Biopharmaceuticals Inc. | Removal of plasmin or plasminogen from cryoprecipitate |
-
1986
- 1986-10-03 JP JP61236720A patent/JPS6391080A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8563288B2 (en) | 2001-05-21 | 2013-10-22 | Omrix Biopharmaceuticals Inc. | Removal of plasmin or plasminogen from cryoprecipitate |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DE68915675T3 (en) | Process for the purification of vitamin K-dependent proteins. | |
| KR20010049991A (en) | Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins by means of affinity chromatography | |
| JPH08259449A (en) | Preparation of pharmaceutical preparation containing hemo-coagulation ix factor | |
| EP0391974B1 (en) | Process for the purification of vitamin k-dependent blood clotting factors | |
| JPH0386900A (en) | Novel thrombin-combining substance and production its | |
| HU219828B (en) | Process for the isolation and purification of vitamin k dependent proteins | |
| JPH02113893A (en) | Method for concentrating one or more of coagulation factors ii, vii, ix, x | |
| US5445958A (en) | Process for purifying blood clotting factors | |
| AU706286B2 (en) | Method for the production of rDSPA alpha1 | |
| JPS6391080A (en) | Purification of human tissue plasminogen activator | |
| EP0276328B1 (en) | Process for separating single-strand human tpa and double-strand human tpa from each other | |
| JPH07508009A (en) | Purification of kringle-containing proteins and especially t-PA | |
| Tamura et al. | Purification of human plasma kallikrein and urokinase by affinity chromatography | |
| JPS61221128A (en) | Monoclonal antibody against plasminogen activator, preparation thereof and method of using same | |
| AU627446C (en) | Chemical process | |
| CA2245554C (en) | Method for the production of rdspa .alpha.1 | |
| JPH064028B2 (en) | Method for producing collagenase | |
| JPH01108985A (en) | Method for refining tissue plasminogen activating factor | |
| JPH01304881A (en) | Production of urokinase precursor | |
| JPH082301B2 (en) | Method for purifying t-PA by reverse phase liquid chromatography | |
| JPS61191699A (en) | Method of purifying mouse interferon | |
| JPS6176419A (en) | Production of purified tissue plasminogen activator | |
| JPS61246131A (en) | Novel type plasminogen activator and preparation thereof | |
| JPS6391400A (en) | Purification of lymphotoxin by hydrophobic chromatography | |
| JPS61234792A (en) | Purification of mouse interferon |