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Molecular engineering method for in vitro evolution of membrane protein

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
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)
公報発行日 令和元年8月7日(2019.8.7)
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
  • 2013JP03767 (2013.6.17) WO
発明の名称 (英語) Molecular engineering method for in vitro evolution of membrane protein
発明の概要(英語) 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.
従来技術、競合技術の概要(英語) Background Art]
As a method of improving an enzyme by evolutionary engineering, a method using liposomes in which a gene library and a cell-free protein synthesis system are enclosed, and a cell sorter has been utilized. In this method, a gene library in which random mutation is introduced into an enzyme gene and a cell-free protein synthesis system are enclosed in liposomes for internal expression of an enzyme. Further, a liposome that contains an enzyme having a higher function is selected by the cell sorter to enable selection of a gene encoding an enzyme having a higher function. By repeating this selection, a gene encoding an enzyme can be evolved (Non Patent Literature 1). This conventional method is solely targeted to soluble proteins.
It is well known that membrane proteins play an important role in functions of cells. Thus, novel molecular evolutionary engineering, particularly enzyme evolutionary engineering, targeting membrane proteins has been required.
[Citation List]
[Non Patent Literature]
[NPL 1] Sunami, T., Sato, K., Matsuura, T., Tsukada, K. , Urabe, I., and Yomo, T. (2006) Analytical biochemistry 357, 128-136
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 has been treated with a nuclease, wherein the nuclease is selected from the group consisting of a ribonuclease and a deoxyribonuclease, and wherein the intraliposomal magnesium concentration is between 28.32 mM and 50 mM.

[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 of claim 1.

[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
(b) a cell-free protein synthesis system, wherein the unilamellar liposome has been treated with a nuclease, wherein the nuclease is a ribonuclease, and wherein the intraliposomal magnesium concentration is between 28.32 mM and 50 mM.

[claim8]
8. The unilamellar liposome of claim 7, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (c) a factor that binds to a ligand transported by the membrane protein.

[claim9]
9. A library comprising a plurality of unilamellar liposomes of claim 7.

[claim10]
10. The library of claim 9, wherein the membrane protein is a transporter, and the unilamellar liposome further comprises (c) a factor that binds to a ligand transported by the membrane protein.

[claim11]
11. A method of producing the unilamellar liposome of claim 1, comprising:
(1) preparing a unilamellar liposome enclosing:
(a) a DNA comprising a promotor 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, and wherein the intraliposomal magnesium concentration is between 28.32 mM and 50 mM.

[claim12]
12. The method of claim 11 wherein the unilamellar liposome further encloses:
(e) a factor that binds to a ligand transported by the membrane protein.

[claim13]
13. The method of claim 11 or 12, wherein the nuclease is a ribonuclease.

[claim14]
14. A method of producing a unilamellar liposome, comprising:
(1) preparing a unilamellar liposome enclosing:
(a) an RNA comprising a translational initiation sequence, and a sequence encoding a membrane protein; and
(b) a cell-free protein synthesis system; and
(2) treating the unilamellar liposome prepared in (1) with a nuclease, wherein the nuclease is a ribonuclease and wherein the intraliposomal magnesium concentration is between 28.32 mM and 50 mM.

[claim15]
15. The method of claim 14, wherein the unilamellar liposome further encloses:
(c) a factor that binds to a ligand that is transported by the membrane protein.

[claim16]
16. A screening method using a library of unilamellar liposomes, comprising:
(i) providing the library of any one of claims 4 to 6;
(ii) selecting a unilamellar liposome having a desired feature from the library;
(iii) amplifying a DNA included in the unilamellar liposome to obtain an amplified DNA; and
(iv) isolating the amplified DNA of (iii).

[claim17]
17. A screening method using a library of unilamellar liposomes, comprising:
(i) providing the library of either claim 9 or claim 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 to obtain a generated DNA;
(iv) amplifying the generated DNA of (iii) to obtain amplified DNA; and
(v) isolating the amplified DNA of (iv).
  • 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 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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