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Molecular engineering method for in vitro evolution of membrane protein NEW

外国特許コード F170009205
整理番号 E102P05WO
掲載日 2017年9月12日
出願国 欧州特許庁(EPO)
出願番号 13810803
公報番号 2876159
出願日 平成25年6月17日(2013.6.17)
公報発行日 平成27年5月27日(2015.5.27)
国際出願番号 JP2013003767
国際公開番号 WO2014002424
国際出願日 平成25年6月17日(2013.6.17)
国際公開日 平成26年1月3日(2014.1.3)
優先権データ
  • 特願2012-145795 (2012.6.28) JP
  • 2013WO-JP03767 (2013.6.17) WO
発明の名称 (英語) Molecular engineering method for in vitro evolution of membrane protein NEW
発明の概要(英語) (EP2876159)
The objective of the present invention is to improve the efficiency of screening/selection of a membrane protein in molecular evolutionary engineering (for example, an enzyme evolutionary method).
The above-described objective is achieved by providing a unilamellar liposome comprising: (a) a DNA comprising a promoter sequence, a translational initiation sequence, and a sequence encoding a membrane protein; (b) an RNA polymerase; (c) a ribonucleotide; and (d) a cell-free protein synthesis system.
In one aspect of the present invention, the membrane protein is a transporter, and the unilamellar liposome further comprises (e) a factor that binds to a ligand transported by the membrane protein.
特許請求の範囲(英語) [claim1]
1. A unilamellar liposome comprising: (a) a DNA comprising a promoter sequence, a translational initiation sequence, and a sequence encoding a membrane protein; (b) an RNA polymerase; (c) a ribonucleotide; and (d) a cell-free protein synthesis system, wherein the unilamellar liposome is treated with a nuclease, and the nuclease is selected from the group consisting of a ribonuclease and a deoxyribonuclease.
[claim2]
2. The unilamellar liposome of claim 1, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (e) a factor that binds to a ligand transported by the membrane protein.
[claim3]
3. The unilamellar liposome of claim 1, wherein the nuclease is a ribonuclease.
[claim4]
4. A library comprising a plurality of unilamellar liposomes, wherein the unilamellar liposome comprises: (a) a DNA comprising a promoter sequence, a translational initiation sequence, and a sequence encoding a membrane protein; (b) an RNA polymerase; (c) a ribonucleotide; and (d) a cell-free protein synthesis system, wherein the unilamellar liposome is treated with a nuclease, and the nuclease is selected from the group consisting of a ribonuclease and a deoxyribonuclease.
[claim5]
5. The library of claim 4, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (e) a factor that binds to a ligand transported by the membrane protein.
[claim6]
6. The library of claim 4, wherein the nuclease is a ribonuclease.
[claim7]
7. A unilamellar liposome comprising: (a) an RNA comprising a translational initiation sequence, and a sequence encoding a membrane protein; and (d) a cell-free protein synthesis system, wherein the unilamellar liposome is treated with a nuclease, and the nuclease is a ribonuclease.
[claim8]
8. The unilamellar liposome of claim 7, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (e) a factor that binds to a ligand transported by the membrane protein.
[claim9]
9. A library comprising a plurality of unilamellar liposomes, wherein the unilamellar liposome comprises: (a) an RNA comprising a translational initiation sequence, and a sequence encoding a membrane protein; and (d) a cell-free protein synthesis system, wherein the unilamellar liposome is treated with a nuclease, and the nuclease is a ribonuclease.
[claim10]
10. The library of claim 9, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (e) a factor that binds to a ligand transported by the membrane protein.
[claim11]
11. A method of producing a unilamellar liposome treated with a nuclease, comprising: (1) preparing a unilamellar liposome enclosing: (a) a DNA comprising a promoter sequence, a translational initiation sequence, and a sequence encoding a membrane protein; (b) an RNA polymerase; (c) a ribonucleotide; and (d) a cell-free protein synthesis system; and(2) treating the unilamellar liposome prepared in (1) with a nuclease, wherein the nuclease is selected from the group consisting of a ribonuclease and a deoxyribonuclease.
[claim12]
12. A method of producing a unilamellar liposome treated with a nuclease, comprising: (1) preparing a unilamellar liposome enclosing: (a) a DNA comprising a promoter sequence, a translational initiation sequence, and a sequence encoding a membrane protein that is a transporter; (b) an RNA polymerase; (c) a ribonucleotide; (d) a cell-free protein synthesis system; and (e) a factor that binds to a ligand transported by the membrane protein; and(2) treating the unilamellar liposome prepared in (1) with a nuclease, wherein the nuclease is selected from the group consisting of a ribonuclease and a deoxyribonuclease.
[claim13]
13. The method of claim 11 or 12, wherein the nuclease is a ribonuclease.
[claim14]
14. A method of producing a unilamellar liposome treated with a nuclease, comprising: (1) preparing a unilamellar liposome enclosing: (a) an RNA comprising a translational initiation sequence, and a sequence encoding a membrane protein; and (d) a cell-free protein synthesis system; and(2) treating the unilamellar liposome prepared in (1) with a nuclease, wherein the nuclease is a ribonuclease.
[claim15]
15. A method of producing a unilamellar liposome treated with a nuclease, comprising: (1) preparing a unilamellar liposome enclosing: (a) an RNA comprising a translational initiation sequence, and a sequence encoding a membrane protein that is a transporter; (d) a cell-free protein synthesis system; and (e) a factor that binds to a ligand that is transported by the membrane protein; and(2) treating the unilamellar liposome prepared in (1) with a nuclease, wherein the nuclease is a ribonuclease.
[claim16]
16. A screening method using a library of unilamellar liposomes, comprising: (i) providing a library of any of claims 4 to 6; (ii) selecting a unilamellar liposome having a desired feature from the library; (iii) amplifying an DNA included in the unilamellar liposome; and (iv) isolating the amplified DNA.
[claim17]
17. A screening method using a library of unilamellar liposomes, comprising: (i) providing a library of claim 9 or 10; (ii) selecting a unilamellar liposome having a desired feature from the library; (iii) generating a DNA by operating a reverse transcriptase on an RNA included in the unilamellar liposome; (iv) amplifying the generated DNA; and (v) isolating the amplified DNA.
  • 出願人(英語)
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • YOMO TETSUYA
  • MATSUURA TOMOAKI
  • SOGA HARUKA
  • WATANABE HAJIME
  • FUJII SATOSHI
国際特許分類(IPC)
指定国 (EP2876159)
Contracting States: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
参考情報 (研究プロジェクト等) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
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