JPH022390A - Novel stimulation factor for human granulocyte macrophage colony - Google Patents
Novel stimulation factor for human granulocyte macrophage colonyInfo
- Publication number
- JPH022390A JPH022390A JP63050871A JP5087188A JPH022390A JP H022390 A JPH022390 A JP H022390A JP 63050871 A JP63050871 A JP 63050871A JP 5087188 A JP5087188 A JP 5087188A JP H022390 A JPH022390 A JP H022390A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- acid sequence
- macrophage colony
- coli
- granulocyte macrophage
- 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
- 210000003714 granulocyte Anatomy 0.000 title abstract description 4
- 210000002540 macrophage Anatomy 0.000 title abstract description 4
- 230000000638 stimulation Effects 0.000 title abstract 3
- 101000746373 Homo sapiens Granulocyte-macrophage colony-stimulating factor Proteins 0.000 claims description 3
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 150000001413 amino acids Chemical group 0.000 abstract description 14
- 241000588724 Escherichia coli Species 0.000 abstract description 11
- 210000004027 cell Anatomy 0.000 abstract description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 6
- 235000001014 amino acid Nutrition 0.000 abstract description 5
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 abstract description 4
- 235000018417 cysteine Nutrition 0.000 abstract description 3
- 208000032839 leukemia Diseases 0.000 abstract description 3
- 108010024636 Glutathione Proteins 0.000 abstract description 2
- 229960003180 glutathione Drugs 0.000 abstract description 2
- 239000013612 plasmid Substances 0.000 abstract description 2
- 150000001945 cysteines Chemical class 0.000 abstract 2
- 208000035473 Communicable disease Diseases 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 230000005934 immune activation Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 11
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 8
- 210000002798 bone marrow cell Anatomy 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 4
- YZCKVEUIGOORGS-NJFSPNSNSA-N Tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 4
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 230000008929 regeneration Effects 0.000 description 4
- 238000011069 regeneration method Methods 0.000 description 4
- 229940104230 thymidine Drugs 0.000 description 4
- 229910052722 tritium Inorganic materials 0.000 description 4
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 4
- 241000699800 Cricetinae Species 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000001332 colony forming effect Effects 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000003760 tallow Substances 0.000 description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- OQALFHMKVSJFRR-UHFFFAOYSA-N dityrosine Chemical compound OC(=O)C(N)CC1=CC=C(O)C(C=2C(=CC=C(CC(N)C(O)=O)C=2)O)=C1 OQALFHMKVSJFRR-UHFFFAOYSA-N 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229960000789 guanidine hydrochloride Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 102000004457 Granulocyte-Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000004221 bone function Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 229910002090 carbon oxide Inorganic materials 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 239000003145 cytotoxic factor Substances 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- DANUORFCFTYTSZ-UHFFFAOYSA-N epinigericin Natural products O1C2(C(CC(C)(O2)C2OC(C)(CC2)C2C(CC(O2)C2C(CC(C)C(O)(CO)O2)C)C)C)C(C)C(OC)CC1CC1CCC(C)C(C(C)C(O)=O)O1 DANUORFCFTYTSZ-UHFFFAOYSA-N 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- DANUORFCFTYTSZ-BIBFWWMMSA-N nigericin Chemical compound C([C@@H]1C[C@H]([C@H]([C@]2([C@@H](C[C@](C)(O2)C2O[C@@](C)(CC2)C2[C@H](CC(O2)[C@@H]2[C@H](C[C@@H](C)[C@](O)(CO)O2)C)C)C)O1)C)OC)[C@H]1CC[C@H](C)C([C@@H](C)C(O)=O)O1 DANUORFCFTYTSZ-BIBFWWMMSA-N 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 239000002265 redox agent Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (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、医薬として有用な新規な構造を有するヒト顆
粒球マクロファージコロニー刺激因子（以下、ヒト顆粒
球マクロファージコロニーＩＩ＋ 激因子をＧＭ−Ｃ３
Ｆと略称する）に関する。Detailed Description of the Invention [Industrial Application Field] The present invention provides human granulocyte-macrophage colony-stimulating factor (hereinafter referred to as human granulocyte-macrophage colony-stimulating factor GM-C3) which has a novel structure useful as a medicine.
(abbreviated as F).
（従来の技術および課題を解決するための手段〕ＧＭ−
Ｃ３Ｆは骨職を刺激して、感染防御、免疫などに重要な
役割を果たす顆粒球マクロファージなど白血球の分化・
増殖を促進するサイト力インの一種であり、その−次構
造はすでに報告されている。（Ｇｏｒｄｏｎ Ｇ、 Ｗ
ｏｎｇ ｅｔ、ａ＋、、 ５ｃｉｅｎｃｅ２２８＋ ８
１０−８１５．（１９８５）；Ａｎｔｏｎｙ ｗ、 Ｂ
ｕｒｇｅｓｓ ｅｔ、ａ＋、。(Conventional technology and means for solving problems) GM-
C3F stimulates bone function and promotes the differentiation and differentiation of white blood cells such as granulocytes and macrophages, which play an important role in infection defense and immunity.
It is a type of cytotoxic protein that promotes proliferation, and its secondary structure has already been reported. (Gordon G, W
ong et, a+,, 5science228+ 8
10-815. (1985); Antony W, B
Urges et, a+,.
Ｂｌｏｏｄ、 ６９４３−５０１９８７）本発明者らは
、遺伝子組換え法により形質転換した大腸菌の菌体内に
産生させたＧＭ−Ｃ３Ｆを単離し、回収するにあたり、
大腸菌内ではＧＭＣ３Ｆが不活性な凝集体として得られ
るため、適当な条件で可溶化還元して直鎖状のＧＭ−Ｃ
Ｓ Ｆとして単離し、これを酸化して２組のジスルフィ
ド結合を有する活性なＧＭ−Ｃ３Ｆを導く過程において
、すでに報告されているものとは異なる新規なＧＭ−Ｃ
３Ｆが得られることを見出し、本発明を完成した。Blood, 6943-501987) The present inventors isolated and recovered GM-C3F produced in the cells of Escherichia coli transformed by genetic recombination.
Since GMC3F is obtained as an inactive aggregate in E. coli, it can be solubilized and reduced under appropriate conditions to form linear GM-C.
In the process of isolating SF and oxidizing it to lead to active GM-C3F having two sets of disulfide bonds, a new GM-C3F different from those already reported was created.
It was discovered that 3F could be obtained, and the present invention was completed.
本発明のＧ Ｍ −ＣＳ Ｆは、第１図記載のアミノ酸
配列（第１番目から第１２８番目まで）で特定されるＮ
末端にメチオニン残基が先行したＧＭ−Ｃ３ＦのＮ末端
からアミノ酸が７個（Ｍｅｔ−Ａｌａ−Ｐｒ。The GM-CSF of the present invention has N
There are seven amino acids from the N-terminus of GM-C3F preceded by a methionine residue (Met-Ala-Pr).
Ａｌａ−Ａｒｇ−５ｅｒ−Ｐｒｏ）除去された、第１図
記載のアミノ酸配列の第８番目から第１２８番目のアミ
ノ酸配列で特定される新規なＧＭ−Ｃ３Ｆである。This is a novel GM-C3F identified by the amino acid sequence from the 8th to the 128th amino acid sequence of the amino acid sequence shown in FIG. 1, which has been removed (Ala-Arg-5er-Pro).
Ｃ以下、本明細書では第１図記載のアミノ酸配列（第１
番目から第１２８番目まで）で特定されるＮ末端にメチ
オニン残基が先行したＧＭ−Ｃ３ＦをＡ型とし、Ａ型Ｇ
Ｍ−Ｃ３ＦのＮ末端からアミノ酸が７個除去された本発
明のＧＭ−Ｃ３ＦをＢ型とする）
本発明のＧＭ−Ｃ３Ｆ Ｂ型は、以下に記す方法によ
り得られる。Below, in this specification, the amino acid sequence shown in FIG.
GM-C3F in which a methionine residue precedes the N-terminus specified by
GM-C3F of the present invention in which seven amino acids are removed from the N-terminus of M-C3F is designated as type B) GM-C3F type B of the present invention can be obtained by the method described below.
第１図記載のＧＭ−Ｃ３ＦをコードするＧＭ−ＣＳＦ遺
伝子を常法により単離し、これを発現する組換え大腸菌
を常法により作成する。The GM-CSF gene encoding GM-C3F shown in FIG. 1 is isolated by a conventional method, and a recombinant E. coli expressing this gene is prepared by a conventional method.
好ましくはヒト白血病細胞０９３７株（ＡＴＣＣＣＲＬ
１５９３）由来のヒトＧＭ−Ｃ３Ｆ遺伝子を有する
プラスミドによって大腸菌に一１２株由来のＳＧ ９
３６株を形質転換し、得られた大腸菌をＬブロス培地等
の適当な培地で常法に従い培養し、ＧＭ−Ｃ３Ｆを産生
させる（ＰＣ？出願ＷＯ３７１０２０６０参照）。ＧＭ
−Ｃ３Ｆは不溶性の凝集体として宿主内に蓄積される。Preferably human leukemia cell line 0937 (ATCC CRL
SG9 derived from 112 strains of Escherichia coli by a plasmid carrying the human GM-C3F gene derived from 1593).
36 strain was transformed, and the resulting E. coli was cultured in a suitable medium such as L broth medium according to a conventional method to produce GM-C3F (see PC application WO37102060). GM
-C3F accumulates in the host as insoluble aggregates.
遠心分離等により培養した菌体を集め、適当な手段（例
えば超音波処理、フレンチプレス処理など）を用いて菌
体を破砕して、遠心分離によって不溶性の凝集体をとり
出す。この凝集体を適当な可溶化剤（例えば高濃度のグ
アニジン塩酸又は尿素）及び、分子内及び分子間に存在
すると思われるジスルフィド結合を切断するための還元
剤（例えば２−メルカプトエタノール）を用いて可溶化
する。還元体として含まれるＣＭ−Ｃ３Ｆをゲルろ過ク
ロマトグラフィーなどの手段によって一次精製する。凝
集体をとり出す段階で０ＭＣ３Ｆに夾雑している宿主由
来のタンパク質や核酸、また、細胞膜成分などのうち可
溶性成分およびＣＭＣ５Ｆと分子量の異なる夾雑成分を
上記の操作により除く。このとき、ＧＭ−Ｃ３Ｆ還元体
の純度は次の再生工程に支障の無い程度に低いことが望
ましく、好ましくは、２０〜５０％程度の純度とする。The cultured bacterial cells are collected by centrifugation or the like, and the bacterial cells are disrupted using an appropriate means (eg, ultrasonication, French press treatment, etc.), and insoluble aggregates are removed by centrifugation. This aggregate is dissolved using an appropriate solubilizing agent (e.g., high concentration guanidine hydrochloride or urea) and a reducing agent (e.g., 2-mercaptoethanol) to cleave disulfide bonds that may exist within and between molecules. Solubilize. CM-C3F contained as a reduced product is primarily purified by means such as gel filtration chromatography. In the step of taking out the aggregate, host-derived proteins and nucleic acids contaminating 0MC3F, soluble components among cell membrane components, and contaminant components having a molecular weight different from that of CMC5F are removed by the above operation. At this time, it is desirable that the purity of the reduced GM-C3F is as low as not to interfere with the next regeneration step, and preferably, the purity is about 20 to 50%.
このようにして得られたＧＭ−Ｃ３Ｆの還元体は適当な
酸化還元系（例えばグルタチオン又はシスティンの酸化
型及び還元型の混合物）を有する溶液中で２ｍのジスル
フィド結合を形成させ、活性なＧＭ−ＣＳＦを再生させ
る。再生後、ＧＭ−Ｃ３Ｆ濃度が低い時には限外ろか等
の方法により０．１ｍｇ／ＩＲ１以上、より好ましくは
０．５ｍｇ／ｍ以上の濃度に濃縮する。この再生溶液に
は、−次槽製によって除き得なかった宿主由来物質の他
、酸化還元剤、キレート剤等の夾雑物が存在するので、
ゲルろ過クロマトグラフィーか、あるいはイオン交換高
速液体クロマトグラフィーを用いて該夾雑物を除去する
。・このようにして得られたＧＭ−Ｃ３ＦＡ型、Ｂ型の
混合物を逆相高速液体クロマトグラフィーにかけ、分離
する。各ＧＭ−Ｃ３Ｆ活性画分を分取し、さらに精製し
、ＧＭ−Ｃ３Ｆの構造を検定することにより、目的のＧ
Ｍ−Ｃ３ＦＢ型を得ることができる。以上の各操作によ
り得られたＧＭ−Ｃ３Ｆ Ｂ型はＡ型より高い好中球
生存維持活性およびハムスター骨髄細胞コロニー形成活
性などの生物活性を有することが認められた。The thus obtained reduced form of GM-C3F is formed into a 2m disulfide bond in a solution containing an appropriate redox system (e.g., a mixture of oxidized and reduced forms of glutathione or cysteine) to form an active GM-C3F. Play the CSF. After regeneration, when the GM-C3F concentration is low, it is concentrated to a concentration of 0.1 mg/IR1 or higher, more preferably 0.5 mg/m or higher, using a method such as an ultrafilter. This regeneration solution contains impurities such as redox agents and chelating agents in addition to host-derived substances that could not be removed by the next bath.
The impurities are removed using gel filtration chromatography or ion exchange high performance liquid chromatography. - The thus obtained mixture of GM-C3FA type and B type is subjected to reverse phase high performance liquid chromatography and separated. By separating each GM-C3F active fraction, further purifying it, and assaying the structure of GM-C3F, the desired G
M-C3FB type can be obtained. GM-C3F type B obtained by the above operations was found to have higher biological activities than type A, such as neutrophil survival maintenance activity and hamster bone marrow cell colony forming activity.
以下実施例により、この発明をさらに詳しく説明する。The present invention will be explained in more detail with reference to Examples below.
ス斯Ｉ津１
ヒト白血病細胞０９３７株 （Ａ Ｔ ＣＣＣＲＬ
１５９３）由来のｈＧＭ−Ｃ３Ｆ遺伝子を有するプラス
ミドによって 大腸菌に一１２株由来のＳＧ９３６株（
ＡＴＣＣ３９２６４）を形質転換し、得られた大腸菌（
ＤＳＭ３４７４）（ＰＣＴ出願賀０８７１０２０６０に
記載の公知菌株）を用いて、アンピシリン、カナマイシ
ンを含むしプロス培地で１４時間、３０’Ｃで培養し、
次に４２°Ｃに温度をあげて誘導をかけ、さらに３時間
焙養した。培養後に、菌体を超音波破砕により破壊し、
１２、ＯＯＯｒｐｍで３０分間遠心分離してペレット化
した。Susichitsu1 Human leukemia cell line 0937 (A T CCCRL
SG936 strain derived from E. coli strain 112 (
ATCC39264) and the obtained E. coli (
DSM3474) (known strain described in PCT application No. 087102060) was cultured at 30'C for 14 hours in Pross medium containing ampicillin and kanamycin.
Next, the temperature was raised to 42°C, induction was applied, and the mixture was further roasted for 3 hours. After culturing, the bacterial cells are destroyed by ultrasonic disruption,
12. Pellet by centrifugation at OOOrpm for 30 minutes.
得られたペレットをＯ，１Ｍ Ｔｒｉ−１１（：１（ｐ
ｆｌ ７．５）で洗浄後、６Ｍグアニジン塩酸及びＩ（
１ｍＭ ２−メルカプトエタノールを含む０．１Ｍ
Ｔｒｉ−１１ＣＩ（ｐＨ７，５）を用いて可溶化し、次
いでセファクリル５２００スーパーフアインカラム （
商標：ファルマシア社製）（２，６ｃｍ Ｘ ９４ｃｏ
＋、 ５００ａｄ樹脂）にかけて４°Ｃでゲルろ過クロ
マトグラフィーを行った。）吉川したＣＭＣ３Ｆ含有溶
液の総蛋白量中のＧＭ−Ｃ３Ｆ純度は４７％であった。The obtained pellet was treated with O, 1M Tri-11 (:1(p
fl 7.5), 6M guanidine hydrochloride and I(
0.1M with 1mM 2-mercaptoethanol
Solubilized using Tri-11CI (pH 7,5) and then loaded onto a Sephacryl 5200 Superfine column (
Trademark: Manufactured by Pharmacia) (2.6cm x 94co)
Gel filtration chromatography was performed at 4°C over 500ad resin). ) The GM-C3F purity in the total protein content of Yoshikawa's CMC3F-containing solution was 47%.
さらにこの画分を４１システィン０．４ｍＭシスチンを
含む０．１Ｍ Ｔｒｉ−ＨＣＩ（ｐｔｌ ７．５）で４
°Ｃ５１２時間透析を行い、さらに新しいバッファー溶
液で４”Ｃ１１晩透析を行って、酸化型ＧＭ−Ｃ３Ｆを
含む再生溶液を得た。この溶液中のＧＭ−Ｃ２Ｆ４度は
０．０５ｍｇ／ ｍｌ であったので、再生溶液を１
２．０００ｒｐ−で３０分間遠心分離後、上清を限外ろ
かにより０．４ｍｇ７ｍ１に濃縮した。 得られた濃縮
液をセファデックスＧ−１００（商標；ファルマシア社
製） （２，６ｃｎ＋Ｘ９４ｃｍ、５００ｍ樹脂）にか
けてゲルろ過クロマトグラフィーを行い、溶出したＧＭ
−Ｃ３Ｆ含有溶液を、再度、限外ろかにより１．０ ｍ
ｇ／ ｍｌに濃縮した。This fraction was further treated with 0.1M Tri-HCI (ptl 7.5) containing 0.4mM cystine.
Dialysis was performed for 12 hours at 5°C, and further dialysis was performed for 11 nights at 4"C against a fresh buffer solution to obtain a regenerated solution containing oxidized GM-C3F. The GM-C2F content in this solution was 0.05 mg/ml. Therefore, the regeneration solution was
After centrifugation at 2.000 rpm for 30 minutes, the supernatant was concentrated to 0.4 mg 7 ml by ultrafiltration. The obtained concentrated solution was subjected to gel filtration chromatography using Sephadex G-100 (trademark; manufactured by Pharmacia) (2.6cn+X94cm, 500m resin), and the eluted GM
-C3F-containing solution was filtered again using an ultrafilter at 1.0 m
g/ml.
このようにして得られた、ＧＭ−Ｃ３Ｆとして１．８８
ｍｇを含む溶液を下記条件によりＨＰＬＣを用いて精製
を行った。1.88 as GM-C3F thus obtained
A solution containing mg was purified using HPLC under the following conditions.
カラム：スミパックス ＯＤＳ Ａ−２１３（１０ｍ
ｍ Ｘ ２５０ｍ翔）（住友化学製）移動相：Ａ液０．
１２％トリフルオロ酢酸水溶液Ｂ液 水
溶出条件ニ一定の割合（Ａ液 ４３％、Ｂ液５７％）で
？８出させる。Column: Sumipax ODS A-213 (10m
m x 250m) (manufactured by Sumitomo Chemical) Mobile phase: A solution 0.
12% trifluoroacetic acid aqueous solution B liquid under certain water elution conditions (liquid A 43%, liquid B 57%)? Let's roll 8.
流量：３ｍｆｌ／ｍｉｎ
カラム温度：室温
検出器：紫外線吸収計（測定波長２３０ｎｍ）保持時間
ｊ（１，Ｍ−Ｃ３Ｆ Ａ型 １７分Ｃ，Ｍ−Ｃ３Ｆ
Ｂ型 ２１分
分取したＧＭ−Ｃ３Ｆ Ａ型 Ｂ型の各成分をＢｒａ
ｄｆｏｒｄらの方法（Ｂｒａｄｆｏｒｄ、 Ｍ、 ｅｔ
、ａｌ、、 Ａｎａｌ。Flow rate: 3 mfl/min Column temperature: Room temperature Detector: Ultraviolet absorption meter (measurement wavelength 230 nm) Retention time j (1, M-C3F Type A 17 minutes C, M-C3F
Type B Each component of GM-C3F type A and type B was fractionated for 21 minutes.
dford et al.'s method (Bradford, M. et al.
,al,, Anal.
Ｂｉｏｃｈｅｍ、ユ２２４８（１９７６））によりタン
パク量を測定した結果、ＧＭ−Ｃ３Ｆ Ａ型０．５４
ｍｇ、 Ｇ Ｍ −Ｃ３Ｆ Ｃ型０．４ｈｇを得た
。As a result of measuring the protein amount using Biochem, Yu2248 (1976)), GM-C3F type A was 0.54.
mg, and 0.4 hg of GM-C3FC type C were obtained.
ＧＭ−Ｃ３Ｆの粗製品の高速液体クロマトグラムを第２
図に示す。The second high-performance liquid chromatogram of the crude product of GM-C3F
As shown in the figure.
尖血班主
実施例１で得たＧＭ−Ｃ３Ｆ Ｂ型について気相アミ
ノ酸シークエンサーを用いてＮ末端アミノ酸配列を分析
した。その結果、ＧＭ−Ｃ３Ｆ Ｂ型はＡ型のＮ末端
アミノ酸が７個（Ｍｅｔ−Ａｌａ−Ｐｒ。The N-terminal amino acid sequence of GM-C3F type B obtained in Example 1 was analyzed using a gas phase amino acid sequencer. As a result, GM-C3F type B has seven N-terminal amino acids of type A (Met-Ala-Pr.
Ａｌａ−Ａｒｇ−５ｅｒ−Ｐｒｏ）欠失したものである
ことが判明した。It was found that the protein had been deleted (Ala-Arg-5er-Pro).
実藷且Ｊ
ハムスター骨髄細胞のコロニー形成活性試験方法
ハムスター大腿骨を無菌的に摘出し、２％牛脂児血清含
有ａ−ＭＥＭ培養液（Ｓｔａｎｎｅｒｓ＋Ｃ，Ｐ、、ｅ
ｔ。J. Hamster Bone Marrow Cell Colony Forming Activity Test Method Hamster femurs were removed aseptically and cultured in a-MEM culture medium containing 2% tallow serum (Stanners+C, P, e
t.
ａｌ、、Ｎａｔｕｒｅ Ｎｅｗ Ｂｉｏｌｏｇｙ、２３
０：５２（１９７２） （Ｆｌｏｗ社製））を注入し、
骨髄細胞を洗い出した。この細胞をピペッティングでば
らばらにし、３分間静置する。細胞浮遊液をとり、同じ
培養液で洗浄した後、４０％牛脂児血清含有α−ＭＥＭ
培養液でｌＸｌ０／ｍ１の細胞濃度に調製する。この骨
髄細胞浮遊液および２％牛脂児血清含有α−ＭＥＭ培養
液で段階的に希釈したヒトＧＭ−Ｃ３Ｆをウェルに１０
０Ｉｌずつ入れて、３７°Ｃ，５％−酸化炭素の条件下
で培養する。培養４２時間後にα−ＭＥＭ培養液で４０
μｃｉ／ｘｉ！に調製したトリチウム標識チミジンを２
５μｌずつ各ウェルに添加しさらに６時間培養後、細胞
に取り込まれた放射活性を測定する。al., Nature New Biology, 23
0:52 (1972) (manufactured by Flow)),
Bone marrow cells were washed out. The cells are broken up by pipetting and allowed to stand for 3 minutes. After taking the cell suspension and washing it with the same culture medium, add α-MEM containing 40% tallow serum.
Adjust the cell concentration to 1X10/ml with culture medium. This bone marrow cell suspension and human GM-C3F serially diluted with α-MEM culture medium containing 2% tallow serum were added to wells for 10
0 Il each and cultured at 37°C under 5% carbon oxide conditions. After 42 hours of culture, incubate with α-MEM culture solution for 40 hours.
μci/xi! Tritium-labeled thymidine prepared in
After adding 5 μl to each well and culturing for a further 6 hours, the radioactivity incorporated into the cells is measured.
ＣＣＶ／ＣＣ）／２のトリチウム標識チミジン取り込み
を誘導するＧＭ−Ｃ３Ｆ量を５０単位／ｒｄと定義して
ＣＭ−Ｃ３ＦＯ比活性を求めた。但し、Ｃｖは最大トリ
チウム標識チミジン取り込み量ＣＣはＧＭ−Ｃ３Ｆ非存
在下に取り込まれたトリチウム標識チミジン量を表わす
。The specific activity of CM-C3FO was determined by defining the amount of GM-C3F that induces the incorporation of tritium-labeled thymidine in CCV/CC)/2 as 50 units/rd. However, Cv represents the maximum amount of tritium-labeled thymidine incorporated; CC represents the amount of tritium-labeled thymidine incorporated in the absence of GM-C3F.
試験結果 表１
表１の結果から、ＧＭ−Ｃ３Ｆ Ｂ型はＧＭ−Ｃ３Ｆ
Ａ型と同様、ハムスター骨髄細胞コロニー形成活性
を有することが認められた。Test results Table 1 From the results in Table 1, GM-C3F type B is GM-C3F
Like type A, it was found to have hamster bone marrow cell colony forming activity.
第１図は組み換え大腸菌由来のＧＭ−Ｃ３Ｆのアミノ酸
配列を示す。
但し、Ａ：アラニン、Ｃニジスティン、Ｄ＝アスパラギ
ン酸、Ｅ：グルタミン酸、Ｆ：フェニルアラニン、Ｇニ
ゲリシン、Ｈ：ヒスチジン、１：イソロイシン、Ｋ：リ
ジン、Ｌ：ロイシン、Ｍ：メチオニン、Ｎ：アスパラギ
ン、Ｐニブロリン、Ｑ：グルタミン、Ｒ：アルギニン、
Ｓ：セリン、Ｔ：スレオニン、ｖ：バリン、ｗニトリブ
トファン、Ｙ二チロシンを各々表す。
第２図は実施例にて用いた粗製品ＧＭ−Ｃ３Ｆの高速液
体クロマトグラムを示す。
第１図
Ｍ Ａ Ｐ Ａ ＲＳ Ｐ Ｓ Ｐ
Ｓ Ｔ Ｑ Ｐ Ｗ Ｅ Ｈｌ ２
３ ４ ５ ６ ７ ８ ９］０１１ １２
１３１４１５＋６Ｖ Ｎ Ａ ｒ Ｑ Ｅ’
Ａ ＲＲＬ Ｌ Ｎ Ｌ Ｓ Ｒ１７
１８１９２０２１２２２３２４２５２６２７２ｇ ２
９ ３０ ３１ＤＴＡＡＥＭＮＥＴＶＥＶ Ｉ
５Ｅ３２ ３３ ３４ ３５ ３６ ３７ ３８ ３９
４０ ４＋ ４２ ４３ ４４ ４５ ４６ＭＦＤ
ＬＱＥＰＴＣＬＱＴＲＬＥ
４７ ４８ ４９ ＥＯ５１５２５３５４５５５６５
７５８５９６０６１し ＹＫＱＧＬＲＧＳＬＴＫＬＫ
Ｇ
ＰＬＴＭＩＡＳＨＹＫＱＨＣＰＰ
ＴＰＥＴＳＣＡＴＱ Ｉ ＩＴＦＥＳ９２ ９３
９４ ９：ｌ ９６ ９７ ９８ ９９１００１０
１１０２１０３１０４１０５１０６ＦＫＥＮＬＫＤＦＬ
ＬＶ Ｉ ＰＦＤ１０７１０８１０９１１０１
１＋ １１２１１３１１４１１５１１６１１７１１８
１１９１２０１２１−Ｓ−Ｓ−結合 ５５番目と９７
７番目！スティン８９番目と１２２５目のンスティンFIG. 1 shows the amino acid sequence of GM-C3F derived from recombinant E. coli. However, A: alanine, C nigistein, D = aspartic acid, E: glutamic acid, F: phenylalanine, G nigericin, H: histidine, 1: isoleucine, K: lysine, L: leucine, M: methionine, N: asparagine, P Nibroline, Q: Glutamine, R: Arginine,
S: serine, T: threonine, v: valine, w nitributophan, Y dityrosine, respectively. FIG. 2 shows a high performance liquid chromatogram of the crude product GM-C3F used in the examples. Figure 1 M A P A R S P S P
S T Q P W E Hl 2
3 4 5 6 7 8 9] 011 12
131415+6V N A r Q E'
A RRL L N L S R17
181920212223242526272g 2
9 30 31DTAAEMNETVEV I
5E32 33 34 35 36 37 38 39
40 4+ 42 43 44 45 46MFD
LQEPTCLQTRLE 47 48 49 EO5152535455565
758596061 YKQGLRGSLTKLK
G PLTMIASHYKQHCPP TPETSCATQ I ITFES92 93
94 9:l 96 97 98 9910010
1102103104105106FKENLKDFL
LV I PFD1071081091101
1+ 112113114115116117118
119120121-SS-bond 55th and 97th
Seventh! Sting 89th and 1225th Sting
Claims (1)
のアミノ酸配列で特定されるヒト顆粒球マクロファージ
コロニー刺激因子ポリペプチドHuman granulocyte-macrophage colony-stimulating factor polypeptide specified by the amino acid sequence from the 8th to the 128th amino acid sequence shown in Figure 1
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63050871A JPH022390A (en) | 1988-02-23 | 1988-03-03 | Novel stimulation factor for human granulocyte macrophage colony |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4163388 | 1988-02-23 | ||
| JP63-41633 | 1988-02-23 | ||
| JP63050871A JPH022390A (en) | 1988-02-23 | 1988-03-03 | Novel stimulation factor for human granulocyte macrophage colony |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH022390A true JPH022390A (en) | 1990-01-08 |
Family
ID=26381278
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63050871A Pending JPH022390A (en) | 1988-02-23 | 1988-03-03 | Novel stimulation factor for human granulocyte macrophage colony |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH022390A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997012054A1 (en) * | 1995-09-28 | 1997-04-03 | Otsuka Pharmaceutical Co., Ltd. | Neutrophil chemotactic lymphokine, and drug and kit for the diagnosis of drug hypersensitive granulocytopenia containing the same |
| US6911204B2 (en) | 2000-08-11 | 2005-06-28 | Favrille, Inc. | Method and composition for altering a B cell mediated pathology |
| US7726941B2 (en) | 2004-07-30 | 2010-06-01 | Vestas Wind Systems A/S | Methods of handling wind turbine blades and mounting said blades on a wind turbine, system and gripping unit for handling a wind turbine blade |
-
1988
- 1988-03-03 JP JP63050871A patent/JPH022390A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1997012054A1 (en) * | 1995-09-28 | 1997-04-03 | Otsuka Pharmaceutical Co., Ltd. | Neutrophil chemotactic lymphokine, and drug and kit for the diagnosis of drug hypersensitive granulocytopenia containing the same |
| US6911204B2 (en) | 2000-08-11 | 2005-06-28 | Favrille, Inc. | Method and composition for altering a B cell mediated pathology |
| US8114404B2 (en) | 2000-08-11 | 2012-02-14 | Mmrglobal, Inc. | Method and composition for altering a B cell mediated pathology |
| US8133486B2 (en) | 2000-08-11 | 2012-03-13 | Mmrglobal, Inc. | Method and composition for altering a B cell mediated pathology |
| US8637638B2 (en) | 2000-08-11 | 2014-01-28 | Mmrglobal, Inc. | Method and composition for altering a B cell mediated pathology |
| US7726941B2 (en) | 2004-07-30 | 2010-06-01 | Vestas Wind Systems A/S | Methods of handling wind turbine blades and mounting said blades on a wind turbine, system and gripping unit for handling a wind turbine blade |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0209539B1 (en) | Homogeneous erythropoietin | |
| Mizel | Studies on the purification and structure-function relationships of murine lymphocyte activating factor (interleukin 1) | |
| US4432895A (en) | Monomeric interferons | |
| KR100771252B1 (en) | Process for the purification of pharmacologically active proteins through cationic exchange chromatography | |
| AU2002366275B2 (en) | Process for the purification and/or isolation of biologically active granulocyte colony stimulating factor | |
| US5196323A (en) | Process for preparing and purifying alpha-interferon | |
| CA1339757C (en) | Production of purified biologically active, bacterially produced recombinant human csf-1 | |
| JPS6261040B2 (en) | ||
| AU624625B2 (en) | Non-glycosylated, recombinant human il2 in the reduced form, the process for obtaining it and its use as a medicament | |
| JP2566919B2 (en) | Method for producing α-interferon | |
| JPH022390A (en) | Novel stimulation factor for human granulocyte macrophage colony | |
| EP0117470A1 (en) | Process for producing interferon (IFN-gamma) subtypes 26K and 21K | |
| Kato et al. | Purification and partial sequence analysis of human interleukin-2 derived from peripheral blood leukocytes | |
| US5391706A (en) | Purification of GM-CSF | |
| KR100531670B1 (en) | Processes for preparing interferon alpha | |
| AU632460B2 (en) | Purification of gm-csf | |
| EP0396555B1 (en) | Purification of monomeric interferon | |
| JPS6226226A (en) | Antitumor polypeptide | |
| EP0446850A2 (en) | Process for purifying recombinant human beta-interferon | |
| KR101113495B1 (en) | A method for purifying recombinant human Interferon beta | |
| KR920009504B1 (en) | Method for purifying inf-alpha | |
| KR920009503B1 (en) | Method for purifying inf-alpha | |
| JPH01165393A (en) | Method for purifying recombinant human erytheropoietin | |
| RU2048521C1 (en) | Method of preparing polypeptide showing human lymphotoxin property | |
| JPS59225195A (en) | Purification of interleukin 2 |