CN104918950A - 亲水性重组蛋白的部分精制方法 - Google Patents
亲水性重组蛋白的部分精制方法 Download PDFInfo
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- CN104918950A CN104918950A CN201380068309.8A CN201380068309A CN104918950A CN 104918950 A CN104918950 A CN 104918950A CN 201380068309 A CN201380068309 A CN 201380068309A CN 104918950 A CN104918950 A CN 104918950A
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- 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
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
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- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Insects & Arthropods (AREA)
- Analytical Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
氨基酸 | HI | 氨基酸 | HI |
丙氨酸(Ala) | 1.8 | 亮氨酸(Leu) | 3.8 |
精氨酸(Arg) | -4.5 | 赖氨酸(Lys) | -3.9 |
天冬酰胺(Asn) | -3.5 | 蛋氨酸(Met) | 1.9 |
天冬氨酸(Asp) | -3.5 | 苯丙氨酸(Phe) | 2.8 |
半胱氨酸(Cys) | 2.5 | 脯氨酸(Pro) | -1.6 |
谷氨酰胺(Gln) | -3.5 | 丝氨酸(Ser) | -0.8 |
谷氨酸(Glu) | -3.5 | 苏氨酸(Thr) | -0.7 |
甘氨酸(Gly) | -0.4 | 色氨酸(Trp) | -0.9 |
组氨酸(His) | -3.2 | 酪氨酸(Tyr) | -1.3 |
异亮氨酸(Ile) | 4.5 | 缬氨酸(Val) | 4.2 |
有机溶剂 | 偶极矩(D) | 有机溶剂 | 偶极矩(D) |
DMSO | 4.3 | NMP | 4.09 |
DMF | 3.86 | 乙腈 | 3.44 |
DMA | 3.72 | 丙酮 | 2.69 |
DMI | 4.05 | 异丙醇 | 1.68 |
样品 | 纯度(%) | |
比较例1 | 超声破碎液 | 19.6 |
实施例1 | 溶解液 | 32.4 |
实施例1 | 溶剂置换后的溶解液 | 76.1 |
实施例1 | 柱洗脱液 | 84.2 |
实施例2 | 实施例3 | 实施例4 | 实施例5 | 实施例6 | 比较例2 | |
溶解用溶剂 | DMSO | DMF | DMA | NMP | DMI | 异丙醇 |
纯度(%) | 29.9 | 23.0 | 26.3 | 22.2 | 21.6 | 9.6 |
Claims (9)
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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JP2012285392 | 2012-12-27 | ||
JP2012-285392 | 2012-12-27 | ||
JP2013-093926 | 2013-04-26 | ||
JP2013093926 | 2013-04-26 | ||
PCT/JP2013/083972 WO2014103847A1 (ja) | 2012-12-27 | 2013-12-18 | 親水性組換えタンパク質の粗精製方法 |
Publications (2)
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CN104918950A true CN104918950A (zh) | 2015-09-16 |
CN104918950B CN104918950B (zh) | 2018-10-12 |
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CN201380068309.8A Active CN104918950B (zh) | 2012-12-27 | 2013-12-18 | 亲水性重组蛋白的部分精制方法 |
Country Status (5)
Country | Link |
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US (1) | US20150344542A1 (zh) |
EP (1) | EP2940033B1 (zh) |
JP (1) | JP6077570B2 (zh) |
CN (1) | CN104918950B (zh) |
WO (1) | WO2014103847A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109912720A (zh) * | 2019-03-14 | 2019-06-21 | 天津大学 | 一种蛛丝蛋白的设计合成方法和纺丝 |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
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JP5782580B2 (ja) | 2013-04-25 | 2015-09-24 | Spiber株式会社 | ポリペプチドヒドロゲル及びその製造方法 |
JP5796147B2 (ja) | 2013-04-25 | 2015-10-21 | Spiber株式会社 | ポリペプチド多孔質体及びその製造方法 |
EP2990414B1 (en) | 2013-04-25 | 2020-12-16 | Spiber Inc. | Polypeptide particle and method for producing same |
US20190225646A1 (en) * | 2016-10-03 | 2019-07-25 | Spiber Inc. | Method for Purifying Recombinant Protein |
US11178934B2 (en) * | 2018-07-18 | 2021-11-23 | Bolt Threads Inc. | Resilin material footwear and fabrication methods |
JPWO2020067572A1 (ja) | 2018-09-28 | 2021-03-18 | Spiber株式会社 | タンパク質組成物の製造方法 |
CN114555108A (zh) | 2019-09-30 | 2022-05-27 | 丝芭博株式会社 | 肌肉组织再生剂 |
CN114502794A (zh) | 2019-09-30 | 2022-05-13 | 丝芭博株式会社 | 蛋白质成型体的制备方法 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1450169A (zh) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | 制备性基因重组蜘蛛拖丝蛋白的分离纯化方法 |
US20100317588A1 (en) * | 2007-11-26 | 2010-12-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Compositions comprising fibrous polypeptides and polysaccharides |
WO2011133886A2 (en) * | 2010-04-23 | 2011-10-27 | Genentech, Inc. | Production of heteromultimeric proteins |
CN102844326A (zh) * | 2010-03-31 | 2012-12-26 | 安西尔克公司 | 不溶性目标蛋白质的分离 |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS61200996A (ja) * | 1985-03-04 | 1986-09-05 | Agency Of Ind Science & Technol | 有機導電体結晶の製造方法 |
DE69131969T2 (de) | 1990-04-20 | 2000-06-15 | The University Of Wyoming, Laramie | Für Spinnenseideprotein kodierende DNS, DNS enthaltender Vektor, transformierte Zelle und Produkte davon |
DE60044057D1 (de) * | 1999-09-03 | 2010-05-06 | Univ Ramot | Verbindungen, zusammensetzungen und verfahren zur behandlung oder vorsorge von alzheimer erkrankung |
US6620917B1 (en) * | 2000-01-20 | 2003-09-16 | The United States Of America As Represented By The Secretary Of The Army | Method for the purification and aqueous fiber spinning of spider silks and other structural proteins |
ATE516304T1 (de) * | 2000-05-23 | 2011-07-15 | Cenes Pharmaceuticals Inc | Nrg-2 nukleinsäuren, polypeptide und diagnostische / therapeutische methoden |
AUPR522501A0 (en) * | 2001-05-25 | 2001-06-14 | Proteome Systems Ltd | Increased solubilisation of hydrophobic proteins |
JP2012012332A (ja) * | 2010-06-30 | 2012-01-19 | Dainippon Sumitomo Pharma Co Ltd | 新規アザインドール誘導体 |
WO2014103846A1 (ja) * | 2012-12-27 | 2014-07-03 | スパイバー株式会社 | 親水性組換えタンパク質の抽出方法 |
-
2013
- 2013-12-18 EP EP13868047.5A patent/EP2940033B1/en active Active
- 2013-12-18 CN CN201380068309.8A patent/CN104918950B/zh active Active
- 2013-12-18 US US14/655,027 patent/US20150344542A1/en not_active Abandoned
- 2013-12-18 WO PCT/JP2013/083972 patent/WO2014103847A1/ja active Application Filing
- 2013-12-18 JP JP2014554365A patent/JP6077570B2/ja active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1450169A (zh) * | 2003-05-08 | 2003-10-22 | 福建师范大学 | 制备性基因重组蜘蛛拖丝蛋白的分离纯化方法 |
US20100317588A1 (en) * | 2007-11-26 | 2010-12-16 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Compositions comprising fibrous polypeptides and polysaccharides |
CN102844326A (zh) * | 2010-03-31 | 2012-12-26 | 安西尔克公司 | 不溶性目标蛋白质的分离 |
WO2011133886A2 (en) * | 2010-04-23 | 2011-10-27 | Genentech, Inc. | Production of heteromultimeric proteins |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109912720A (zh) * | 2019-03-14 | 2019-06-21 | 天津大学 | 一种蛛丝蛋白的设计合成方法和纺丝 |
CN109912720B (zh) * | 2019-03-14 | 2021-12-07 | 天津大学 | 一种蛛丝蛋白的设计合成方法和纺丝 |
Also Published As
Publication number | Publication date |
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WO2014103847A1 (ja) | 2014-07-03 |
JPWO2014103847A1 (ja) | 2017-01-12 |
US20150344542A1 (en) | 2015-12-03 |
EP2940033A4 (en) | 2016-10-12 |
EP2940033B1 (en) | 2018-04-25 |
CN104918950B (zh) | 2018-10-12 |
EP2940033A1 (en) | 2015-11-04 |
JP6077570B2 (ja) | 2017-02-08 |
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