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Method for producing membrane protein UPDATE_EN achieved

Foreign code F190009892
File No. 07366-EP
Posted date Aug 26, 2019
Country EPO
Application number 08846826
Gazette No. 2213747
Date of filing Nov 5, 2008
Gazette Date Aug 4, 2010
International application number JP2008070116
International publication number WO2009060857
Date of international filing Nov 5, 2008
Date of international publication May 14, 2009
Priority data
  • P2007-286899 (Nov 5, 2007) JP
  • 2008JP70116 (Nov 5, 2008) WO
Title Method for producing membrane protein UPDATE_EN achieved
Abstract A method is provided for producing a membrane protein folded to its native structure or active structure in a lipid disc or a liposome. The method comprises the following steps: (a) preparing a reaction solution for cell-free protein synthesis containing a polynucleotide encoding a membrane protein, a steroidal detergent, and a phospholipid, wherein the steroidal detergent is contained at a concentration higher than its critical micelle concentration; (b) decreasing the concentration of said steroidal detergent in the reaction solution; and (c) synthesizing the membrane protein simultaneously with formation of a lipid disk or liposome into which the synthesized membrane protein is integrated.
Outline of related art and contending technology BACKGROUND ART
Primary structures of proteins encoded by genome in various organisms have been revealed based on results of various genome projects. Approximately 30 % of proteins in higher organisms have been presumed to be an endogenous membrane protein (integral membrane protein) having transmembrane helix. Membrane proteins are involved in signal transduction, mass transportation, energy production, formation of cytoskeleton etc. at cell membranes. In addition, the membrane protein is very important as a potential drug target. Actually, it is known that approximately 70 % of commercially available pharmaceutical agents act on membrane proteins, especially on a G protein-coupled receptor. Protein 3000 Project in Japan which was conducted in consideration of the importance of protein structures has revealed stereostructures of more proteins than was initially expected and produced internationally great results. However, there are merely a few cases that have revealed the stereostructures of membrane proteins, since it is difficult to produce crystals of membrane proteins suitable for structural analysis.
The applicant of the present application has already developed a method for producing an insoluble protein using a cell-free protein synthesis system in the presence of detergent (surfactant) without the protein being insolubilized (see, for example, Patent Document 1 and Non-Patent Document 1). This method can be merely applied to a case where various membrane proteins can be abundantly synthesized as soluble fraction and a case where a detergent used does not inhibit protein synthesis. Accordingly, there are some detergents that cannot be selected for the method as those which contribute to the stabilization of the synthesized protein.
Moreover, a method for synthesizing a membrane protein having a biological function by merely adding a lipid liposome to a cell-free protein synthesis system is also reported (see Patent Document 2 and Non-Patent Document 2). However, it is considered that the method is not necessarily versatile, since the introducibility of membrane proteins into a liposome depends on their respective properties and the probability of contact between a synthesized polypeptide chain and a liposome is low.
[Patent Document 1] Japanese Patent Kokai Publication No. 2003-18999A
[Patent Document 2] Japanese Patent Kokai Publication No. 2005-225796A
[Non-Patent Document 1] Ishihara G. et al., (2005) Protein Expression and Purification, Vol. 41, pp.27-37
[Non-Patent Document 2] Kalmbach, R. et al., (2007) Journal of Molecular Biology, Vol. 371, pp. 639-648
Scope of claims [claim1]
1. A method for producing a membrane protein folded to its native or active structure in a lipid disk or liposome, comprising the following steps:
(a) preparing a reaction solution for cell-free protein synthesis containing a polynucleotide encoding a membrane protein, a steroidal detergent, and a phospholipid, wherein the steroidal detergent is contained at a concentration higher than its critical micelle concentration,
(b) decreasing the concentration of said steroidal detergent in the reaction solution, and
(c) synthesizing the membrane protein simultaneously with formation of a lipid disk or liposome into which the synthesized membrane protein is integrated.

[claim2]
2. The method of claim 1, wherein said membrane protein is synthesized by a cell-free protein synthesis reaction using a dialysis method, and said steroidal detergent is contained in the reaction solution at an initial concentration of 1.5 to 10 times higher than its critical micelle concentration.

[claim3]
3. The method of claim 1 or 2, wherein said steroidal detergent is digitonin, cholate or CHAPS.

[claim4]
4. The method of claim 1 or 2, wherein said reaction solution for cell-free protein synthesis contains 10 to 20mg/ml sodium cholate at an initial concentration.

[claim5]
5. The method of claim 1 or 2, wherein said reaction solution for cell-free protein synthesis contains 5 to 10mg/mL CHAPS at an initial concentration.

[claim6]
6. The method of claim 1 or 2, wherein said reaction solution for cell-free protein synthesis contains 1 to 4mg/mL digitonin at an initial concentration.

[claim7]
7. The method of claim 1, wherein said step (b) comprises adsorbing the detergent by using a resin capable of binding to the detergent.

[claim8]
8. The method of any one of claims 1 to 7, wherein the membrane protein comprises a receptor protein, a channel protein, a transporter, a membrane-bound enzyme, or a partial sequence, a homologous sequence, a modified sequence and an inducible sequence thereof.

[claim9]
9. The method of any one of claims 1 to 8, further comprising a step of solubilizing the membrane protein from the (resultant) complex with the lipid disk or liposome to purify the membrane protein.

[claim10]
10. A composition for screening a pharmaceutical agent, the composition comprising the membrane protein produced by the method of any one of claims 1 to 8, the protein being in a state of being integrated into a lipid disk or liposome.

[claim11]
11. A composition for delivering a pharmaceutical agent comprising the membrane protein produced by the method of any one of claims 1 to 8 and a biologically active agent, both of which are in a state of being integrated into a lipid disk or liposome.
  • Applicant
  • RIKEN
  • Inventor
  • YOKOYAMA, Shigeyuki
  • SHIMONO, Kazumi
  • SHIROUZU, Mikako
  • GOTO, Mie
IPC(International Patent Classification)
Specified countries Contracting States: AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MT NL NO PL PT RO SE SI SK TR

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