Top > Search of International Patents > Molecular engineering method for in vitro evolution of membrane protein

Molecular engineering method for in vitro evolution of membrane protein UPDATE_EN

Foreign code F170009205
File No. E102P05WO
Posted date Sep 12, 2017
Country EPO
Application number 13810803
Gazette No. 2876159
Gazette No. 2876159
Date of filing Jun 17, 2013
Gazette Date May 27, 2015
Gazette Date Aug 7, 2019
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
  • 2013JP03767 (Jun 17, 2013) WO
Title Molecular engineering method for in vitro evolution of membrane protein UPDATE_EN
Abstract 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.
Outline of related art and contending technology 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
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 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).
  • Applicant
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • Inventor
  • YOMO, Tetsuya
  • MATSUURA, Tomoaki
  • SOGA, Haruka
  • WATANABE, Hajime
  • FUJII, Satoshi
IPC(International Patent Classification)
Specified countries 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
Reference ( R and D project ) ERATO YOMO Dynamical Micro-scale Reaction Environment AREA
Please contact us by E-mail or facsimile if you have any interests on this patent.

PAGE TOP

close
close
close
close
close
close