ATE342367T1 - Verwendung von fusionsproteinen, deren n- terminaler teil aus einem hirudinderivat besteht, zur herstellung rekombinanter proteine durch sekretion durch hefen - Google Patents

Verwendung von fusionsproteinen, deren n- terminaler teil aus einem hirudinderivat besteht, zur herstellung rekombinanter proteine durch sekretion durch hefen

Info

Publication number: ATE342367T1
Authority: AT; Austria
Prior art keywords: nucleic acid; yeast; derivative; acid sequence; proteins
Prior art date: 2001-02-20

Application number

AT02719782T

Other languages

English (en)

Inventor

Paul Habermann

Original Assignee

Sanofi Aventis Deutschland

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.)

2001-02-20

Filing date

2002-02-08

Publication date

2006-11-15

2002-02-08 Application filed by Sanofi Aventis Deutschland filed Critical Sanofi Aventis Deutschland

2006-11-15 Application granted granted Critical

2006-11-15 Publication of ATE342367T1 publication Critical patent/ATE342367T1/de

Links

240000004808 Saccharomyces cerevisiae Species 0.000 title abstract 2
102000007056 Recombinant Fusion Proteins Human genes 0.000 title 1
108010008281 Recombinant Fusion Proteins Proteins 0.000 title 1
102000037865 fusion proteins Human genes 0.000 title 1
108020001507 fusion proteins Proteins 0.000 title 1
230000028327 secretion Effects 0.000 title 1
150000007523 nucleic acids Chemical group 0.000 abstract 5
108020004705 Codon Proteins 0.000 abstract 4
108091028043 Nucleic acid sequence Proteins 0.000 abstract 4
WQPDUTSPKFMPDP-OUMQNGNKSA-N hirudin Chemical compound C([C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC(OS(O)(=O)=O)=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H]1NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H]2CSSC[C@@H](C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@H](C(NCC(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N2)=O)CSSC1)C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)CSSC1)C(C)C)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 WQPDUTSPKFMPDP-OUMQNGNKSA-N 0.000 abstract 3
108090000623 proteins and genes Proteins 0.000 abstract 3
102000004169 proteins and genes Human genes 0.000 abstract 3
239000000126 substance Substances 0.000 abstract 3
239000004475 Arginine Substances 0.000 abstract 2
102000007625 Hirudins Human genes 0.000 abstract 2
108010007267 Hirudins Proteins 0.000 abstract 2
ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 abstract 2
229940006607 hirudin Drugs 0.000 abstract 2
239000004472 Lysine Substances 0.000 abstract 1
KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 abstract 1
102000001708 Protein Isoforms Human genes 0.000 abstract 1
108010029485 Protein Isoforms Proteins 0.000 abstract 1
108010076504 Protein Sorting Signals Proteins 0.000 abstract 1
125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 abstract 1
108020004707 nucleic acids Proteins 0.000 abstract 1
102000039446 nucleic acids Human genes 0.000 abstract 1
239000013612 plasmid Substances 0.000 abstract 1
239000013598 vector Substances 0.000 abstract 1

Classifications

- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide

Landscapes

Health & Medical Sciences (AREA)
Life Sciences & Earth Sciences (AREA)
Chemical & Material Sciences (AREA)
Genetics & Genomics (AREA)
Organic Chemistry (AREA)
Engineering & Computer Science (AREA)
Molecular Biology (AREA)
Zoology (AREA)
Biomedical Technology (AREA)
Biochemistry (AREA)
Biophysics (AREA)
General Health & Medical Sciences (AREA)
General Engineering & Computer Science (AREA)
Biotechnology (AREA)
Wood Science & Technology (AREA)
Bioinformatics & Cheminformatics (AREA)
Proteomics, Peptides & Aminoacids (AREA)
Gastroenterology & Hepatology (AREA)
Mycology (AREA)
Medicinal Chemistry (AREA)
Physics & Mathematics (AREA)
Plant Pathology (AREA)
Microbiology (AREA)
Diabetes (AREA)
Endocrinology (AREA)
Toxicology (AREA)
Tropical Medicine & Parasitology (AREA)
Preparation Of Compounds By Using Micro-Organisms (AREA)
Peptides Or Proteins (AREA)
Micro-Organisms Or Cultivation Processes Thereof (AREA)
Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

AT02719782T 2001-02-20 2002-02-08 Verwendung von fusionsproteinen, deren n- terminaler teil aus einem hirudinderivat besteht, zur herstellung rekombinanter proteine durch sekretion durch hefen ATE342367T1 (de)

Applications Claiming Priority (1)

Application Number	Priority Date	Filing Date	Title
DE10108211A DE10108211A1 (de)	2001-02-20	2001-02-20	Verwendung von Fusionsproteinen, deren N-terminaler Anteil aus einem Hirudinderivat besteht, zur Herstellung rekombinanter Proteine über Sekretion durch Hefen

Publications (1)

Publication Number	Publication Date
ATE342367T1 true ATE342367T1 (de)	2006-11-15

Family

ID=7674912

Family Applications (1)

Application Number	Title	Priority Date	Filing Date
AT02719782T ATE342367T1 (de)	2001-02-20	2002-02-08	Verwendung von fusionsproteinen, deren n- terminaler teil aus einem hirudinderivat besteht, zur herstellung rekombinanter proteine durch sekretion durch hefen

Country Status (21)

Country	Link
US (1)	US20030176673A1 (de)
EP (1)	EP1364032B1 (de)
JP (1)	JP4199543B2 (de)
KR (1)	KR100858833B1 (de)
CN (1)	CN1279171C (de)
AT (1)	ATE342367T1 (de)
AU (1)	AU2002250903B2 (de)
BR (1)	BR0207379A (de)
CA (1)	CA2439042C (de)
CY (1)	CY1106274T1 (de)
DE (2)	DE10108211A1 (de)
DK (1)	DK1364032T3 (de)
ES (1)	ES2272696T3 (de)
IL (2)	IL157416A0 (de)
MX (1)	MXPA03007116A (de)
NO (1)	NO331376B1 (de)
NZ (1)	NZ527642A (de)
PE (1)	PE20020857A1 (de)
PT (1)	PT1364032E (de)
WO (1)	WO2002070722A1 (de)
ZA (1)	ZA200305868B (de)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
US7638618B2 (en) *	2001-02-20	2009-12-29	Sanofi-Aventis Deutschland Gmbh	Nucleic acids encoding a hirudin and pro-insulin as superscretable peptides and for parallel improvement of the exported forms of one or more polypeptides of interest
DE102006031962A1 (de)	2006-07-11	2008-01-17	Sanofi-Aventis Deutschland Gmbh	Amidiertes Insulin Glargin
DE102006031955A1 (de)	2006-07-11	2008-01-17	Sanofi-Aventis Deutschland Gmbh	Verfahren zur Herstellung von Insulinanaloga mit dibasischem B-Kettenende
JP5695909B2 (ja)	2008-01-09	2015-04-08	サノフィ−アベンティス・ドイチュラント・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング	極度に遅延した時間作用プロファイルを有する新規なインスリン誘導体
DE102008003566A1 (de)	2008-01-09	2009-07-16	Sanofi-Aventis Deutschland Gmbh	Neue Insulinderivate mit extrem verzögertem Zeit-/ Wirkungsprofil
DE102008025007A1 (de)	2008-05-24	2009-11-26	Sanofi-Aventis Deutschland Gmbh	Neue Insulinderivate mit extrem verzögertem Zeit-/ Wirkungsprofil
MY152979A (en)	2008-01-09	2014-12-15	Sanofi Aventis Deutschland	Novel insulin derivatives having an extremely delayed time-action profile
RS59913B1 (sr)	2008-10-17	2020-03-31	Sanofi Aventis Deutschland	Kombinacija insulina i glp-1-agonista
CN102596175A (zh)	2009-07-06	2012-07-18	赛诺菲-安万特德国有限公司	含有甲硫氨酸的水性胰岛素制备物
PT3345593T (pt)	2009-11-13	2023-11-27	Sanofi Aventis Deutschland	Composição farmacêutica compreendendo despro36exendina- 4(1-39)-lys6-nh2 e metionina
MY180661A (en)	2009-11-13	2020-12-04	Sanofi Aventis Deutschland	Pharmaceutical composition comprising a glp-1 agonist, an insulin and methionine
PL2611458T3 (pl)	2010-08-30	2017-02-28	Sanofi-Aventis Deutschland Gmbh	Zastosowanie AVE0010 do produkcji leku do leczenia cukrzycy typu 2
US9821032B2 (en)	2011-05-13	2017-11-21	Sanofi-Aventis Deutschland Gmbh	Pharmaceutical combination for improving glycemic control as add-on therapy to basal insulin
AR087693A1 (es)	2011-08-29	2014-04-09	Sanofi Aventis Deutschland	Combinacion farmaceutica para uso en el control glucemico en pacientes con diabetes de tipo 2
TWI559929B (en)	2011-09-01	2016-12-01	Sanofi Aventis Deutschland	Pharmaceutical composition for use in the treatment of a neurodegenerative disease
EP3229828B1 (de)	2014-12-12	2023-04-05	Sanofi-Aventis Deutschland GmbH	Insulin-glargin/lixisenatid-fixierte verhältnisformulierung
TWI748945B (zh)	2015-03-13	2021-12-11	德商賽諾菲阿凡提斯德意志有限公司	第２型糖尿病病患治療
TW201705975A (zh)	2015-03-18	2017-02-16	賽諾菲阿凡提斯德意志有限公司	第２型糖尿病病患之治療

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
CA1341417C (fr) *	1984-03-27	2003-01-21	Paul Tolstoshev	Vecteurs d'expression de l'hirudine, cellules transformees et procede de preparation de l'hirudine
US5705355A (en) *	1984-03-27	1998-01-06	Transgene, S.A.	Hirudin, pharmaceutical compositions comprising it and their use
DE3738541A1 (de) *	1987-11-13	1989-05-24	Hoechst Ag	Verfahren zur isolierung und reinigung von hirudin
US6875589B1 (en) *	1988-06-23	2005-04-05	Hoechst Aktiengesellschaft	Mini-proinsulin, its preparation and use
GB8927722D0 (en) *	1989-12-07	1990-02-07	British Bio Technology	Proteins and nucleic acids
DE4009268A1 (de) *	1990-03-22	1991-09-26	Consortium Elektrochem Ind	Sekretion von hirudinderivaten
ES2093717T3 (es) *	1990-11-08	1997-01-01	Japan Energy Corp	Mutante de hirudina, su produccion, anticoagulante, vector secretor, microorganismo transformado por el indicado vector y produccion de un producto a partir de dicho microorganismo.
FI92601C (fi) *	1992-03-11	1994-12-12	Marja Makarow	Menetelmä hyötyproteiinien erittämiseksi hiivoista
DE19543737A1 (de) *	1995-11-24	1997-05-28	Hoechst Ag	Verfahren zur Ultrafiltration von Peptide oder Proteine enthaltender biologischer Matrices
DE19544233A1 (de) *	1995-11-28	1997-06-05	Hoechst Ag	Verfahren zur Nutzung des Hefe-ADH II-Promotorsystems zur biotechnologischen Produktion heterologer Proteine in hohen Ausbeuten
ATE241695T1 (de) *	1996-03-13	2003-06-15	Delta Biotechnology Ltd	Gärungs-steuerungssysteme
DE10033195A1 (de) *	2000-07-07	2002-03-21	Aventis Pharma Gmbh	Bifunktionale Fusionsproteine aus Hirudin und TAP
US7202059B2 (en) *	2001-02-20	2007-04-10	Sanofi-Aventis Deutschland Gmbh	Fusion proteins capable of being secreted into a fermentation medium

2001
- 2001-02-20 DE DE10108211A patent/DE10108211A1/de not_active Withdrawn
2002
- 2002-02-06 PE PE2002000082A patent/PE20020857A1/es not_active Application Discontinuation
- 2002-02-08 CN CNB028068491A patent/CN1279171C/zh not_active Expired - Fee Related
- 2002-02-08 WO PCT/EP2002/001308 patent/WO2002070722A1/en not_active Ceased
- 2002-02-08 KR KR1020037010932A patent/KR100858833B1/ko not_active Expired - Fee Related
- 2002-02-08 AU AU2002250903A patent/AU2002250903B2/en not_active Ceased
- 2002-02-08 JP JP2002570745A patent/JP4199543B2/ja not_active Expired - Fee Related
- 2002-02-08 CA CA2439042A patent/CA2439042C/en not_active Expired - Fee Related
- 2002-02-08 DE DE60215315T patent/DE60215315T2/de not_active Expired - Lifetime
- 2002-02-08 PT PT02719782T patent/PT1364032E/pt unknown
- 2002-02-08 EP EP02719782A patent/EP1364032B1/de not_active Expired - Lifetime
- 2002-02-08 ES ES02719782T patent/ES2272696T3/es not_active Expired - Lifetime
- 2002-02-08 AT AT02719782T patent/ATE342367T1/de active
- 2002-02-08 MX MXPA03007116A patent/MXPA03007116A/es active IP Right Grant
- 2002-02-08 BR BR0207379-0A patent/BR0207379A/pt not_active Application Discontinuation
- 2002-02-08 NZ NZ527642A patent/NZ527642A/en not_active IP Right Cessation
- 2002-02-08 DK DK02719782T patent/DK1364032T3/da active
- 2002-02-08 IL IL15741602A patent/IL157416A0/xx unknown
- 2002-02-19 US US10/076,632 patent/US20030176673A1/en not_active Abandoned
2003
- 2003-07-30 ZA ZA2003/05868A patent/ZA200305868B/en unknown
- 2003-08-14 IL IL157416A patent/IL157416A/en not_active IP Right Cessation
- 2003-08-19 NO NO20033672A patent/NO331376B1/no not_active IP Right Cessation
2006
- 2006-11-30 CY CY20061101726T patent/CY1106274T1/el unknown

Also Published As

Publication number	Publication date
US20030176673A1 (en)	2003-09-18
NZ527642A (en)	2005-03-24
EP1364032B1 (de)	2006-10-11
HK1065068A1 (en)	2005-02-08
DK1364032T3 (da)	2007-02-19
NO20033672L (no)	2003-10-17
CN1526021A (zh)	2004-09-01
KR100858833B1 (ko)	2008-09-17
MXPA03007116A (es)	2003-11-18
PE20020857A1 (es)	2002-11-04
CA2439042C (en)	2013-06-18
ZA200305868B (en)	2005-02-23
CY1106274T1 (el)	2011-10-12
NO331376B1 (no)	2011-12-12
BR0207379A (pt)	2004-06-15
NO20033672D0 (no)	2003-08-19
WO2002070722A1 (en)	2002-09-12
JP2004518446A (ja)	2004-06-24
DE10108211A1 (de)	2002-08-22
EP1364032A1 (de)	2003-11-26
AU2002250903B2 (en)	2006-12-07
IL157416A (en)	2009-08-03
ES2272696T3 (es)	2007-05-01
KR20030074841A (ko)	2003-09-19
CA2439042A1 (en)	2002-09-12
PT1364032E (pt)	2007-01-31
DE60215315D1 (de)	2006-11-23
DE60215315T2 (de)	2007-05-24
JP4199543B2 (ja)	2008-12-17
IL157416A0 (en)	2004-03-28
CN1279171C (zh)	2006-10-11

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Publication	Publication Date	Title
ATE342367T1 (de)	2006-11-15	Verwendung von fusionsproteinen, deren n- terminaler teil aus einem hirudinderivat besteht, zur herstellung rekombinanter proteine durch sekretion durch hefen
Vedvick et al.	1991	High-level secretion of biologically active aprotinin from the yeast Pichia pastoris
Lind et al.	1995	Novel forms of B‐domain‐deleted recombinant factor VIII molecules: construction and biochemical characterization
KR100227167B1 (ko)	1999-10-15	인체혈청알부민의 n-말단 단편을 함유하는 융합단백질
EP0322094B1 (de)	1992-12-02	N-terminale Fragmente von menschlichem Serumalbumin
AU619553B2 (en)	1992-01-30	Aprotinin analogues and a process for the production thereof
EP0399666A1 (de)	1990-11-28	N-terminale Fragmente von menschliches Serumalbumin enthaltenden Fusionsproteinen
KR880701105A (ko)	1988-07-25	신규 응혈촉진 단백질
DK0755447T3 (da)	2003-10-27	Rekombinante proteiner af filamentøst hæmagglutinin fra Bordetella, især Bordetella Pertussis, fremgangsmåde til fremstilling heraf samt anvendelse heraf i fremstillingen af fremmede proteiner eller vacciner
ATE63757T1 (de)	1991-06-15	Expression durch verwendung von fusionsgenen fuer proteinproduktion.
ATE527364T1 (de)	2011-10-15	Polypeptide erhaltend eine modifizierte menschliche serum-albumin sekretions-pre-sequenz mit erhöhtem ausscheidungsertrag
JP2006501818A5 (de)	2006-08-03
ATE364703T1 (de)	2007-07-15	Herstellung von proteinen aus pro-proteinen durch fusionsproteine abgeleitet von furin oder furinanalogen
DE60209883D1 (de)	2006-05-11	Übersekretierte peptide, deren herstellung, und gleichzeitige verbesserung der sekretierten form eines oder mehrerer anderer polypeptide
Pohlig et al.	1996	Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae
Degryse et al.	1992	Addition of a dipeptide spacer significantly improves secretion of ovine trophoblast interferon in yeast
Okabayashi et al.	1996	Secretory production of recombinant urokinase-type plasminogen activator-annexin V chimeras in Pichia pastoris
Olins et al.	1987	Phosphorylation of high‐and low‐molecular‐mass atrial natriuretic peptide analogs by cyclic AMP‐dependent protein kinase
Chow et al.	1990	Expression of recombinant human glucose-dependent insulinotropic polypeptide in Escherichia coli by sequence-specific proteolysis of a protein A fusion protein
CA2171557C (en)	2007-02-06	A secretion sequence for the production of a heterologous protein in yeast
Baron et al.	1990	Towards the structure of mosaic proteins: Use of protein expression and NMR techniques
Human	2013	Construction of pPIC9 Recombinant Vector Containing Human Stem Cell Factor
Charpigny et al.	0	Addition of
JPH0731496A (ja)	1995-02-03	血管透過性因子の製造方法