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In vitro membrane protein molecular evolutionary engineering technique

Foreign code F170009184
File No. E102P05WO
Posted date Sep 12, 2017
Country United States of America
Application number 201314411892
Gazette No. 20150176004
Date of filing Jun 17, 2013
Gazette Date Jun 25, 2015
International application number JP2013003767
International publication number WO2014002424
Date of international filing Jun 17, 2013
Date of international publication Jan 3, 2014
Priority data
  • P2012-145795 (Jun 28, 2012) JP
  • 2013WO-JP03767 (Jun 17, 2013) WO
Title In vitro membrane protein molecular evolutionary engineering technique
Abstract (US20150176004)
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.
Scope of claims [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 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 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 (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 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 the unilamellar liposome of claim 2, 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 the unilamellar liposome of claim 7, 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.
[claim15]
15. A method of producing the unilamellar liposome of claim 8, 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;
(b) a cell-free protein synthesis system; and
(c) 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 the library of any one 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 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).
  • Inventor, and Inventor/Applicant
  • YOMO TETSUYA
  • MATSUURA TOMOAKI
  • SOGA HARUKA
  • WATANABE HAJIME
  • FUJII SATOSHI
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
IPC(International Patent Classification)
Reference ( R and D project ) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
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