Tools and methods for expression of membrane proteins

Abstract

The disclosure relates cells or cellular systems that express both a membrane protein and a binding domain directed to the membrane protein. Also, methods are provided that use such cells or cellular systems to produce higher amounts of the membrane proteins. Further, the cells or cellular systems can be used as tools for the structural and functional characterization of membrane proteins, as well as for screening and drug discovery efforts targeting membrane proteins.

What is claimed is: 
     
         1 . A method of screening for compounds that bind a complex, the method comprising:
 mixing a test compound with a host cell or a membrane preparation of the host cell; and   measuring binding of the test compound to a complex of a membrane protein and an immunoglobulin single variable domain;   wherein the host cell comprises:
 a first exogenous polynucleotide encoding the membrane protein, 
 a second exogenous polynucleotide encoding an immunoglobulin single variable domain that specifically binds a conformational epitope of the membrane protein, and 
 the membrane associated complex; 
 wherein the expression of each exogenous polynucleotide is under the control of a promoter; 
 wherein the amount of the membrane protein in the recombinant cell is increased by at least two-fold as compared to an otherwise identical control cell not comprising and expressing the second exogenous polynucleotide; 
 wherein the immunoglobulin single variable domain was determined to specifically bind the conformational epitope of the membrane protein and increase the amount of the membrane protein in the host cell by at least two-fold as compared to an otherwise identical host cell not comprising the immunoglobulin single variable domain. 
   
     
     
         2 . The method according to  claim 1 , wherein the immunoglobulin single variable domain was determined to specifically binds an intracellular conformational epitope of the membrane protein by a method comprising:
 detecting specific binding of the immunoglobulin single variable domain to a conformational epitope of the membrane protein;   co-expressing the immunoglobulin single variable domain with the membrane protein in a host cell; and   detecting an increased amount of the membrane protein by at least two-fold in the host cell as compared to an otherwise identical host cell not comprising the immunoglobulin single variable domain.   
     
     
         3 . The method according to  claim 1 , wherein the membrane protein is a GPCR. 
     
     
         4 . The method according to  claim 3 , wherein the immunoglobulin single variable domain specifically binds to an intracellular conformational epitope of the GPCR. 
     
     
         5 . The method according to  claim 1 , wherein detecting specific binding of the immunoglobulin single variable domain to the membrane protein comprises screening a library of immunoglobulin single variable domains to identify an immunoglobulin single variable domain that, when coexpressed with the membrane protein, specifically binds to a conformational epitope in the membrane protein, and increases the amount of the membrane protein in the recombinant cell as compared to an otherwise identical cell not comprising the immunoglobulin single variable domain. 
     
     
         6 . The method according to  claim 5 , wherein screening a library of immunoglobulin single variable domains to identify an immunoglobulin single variable domain comprises identifying an immunoglobulin single variable domain that increases the amount of the membrane protein in the cell by at least two-fold as compared to an otherwise identical cell not comprising the immunoglobulin single variable domain. 
     
     
         7 . The method according to  claim 1 , wherein said promoter is a constitutive promoter or an inducible promoter. 
     
     
         8 . The method according to  claim 1 , wherein the membrane protein and immunoglobulin single variable domain are co-expressed by the host cell. 
     
     
         9 . The method according to  claim 1 , wherein the membrane protein and/or the immunoglobulin single variable domain are operably linked to one or more subcellular targeting sequences. 
     
     
         10 . The method according to  claim 1 , wherein the immunoglobulin single variable domain comprises a peptide comprising four framework regions and three complementary determining regions, or any suitable fragment thereof. 
     
     
         11 . The method according to  claim 10  wherein the immunoglobulin single variable domain is a nanobody. 
     
     
         12 . The recombinant cell of  claim 1 , wherein said immunoglobulin single variable domain stabilizes the membrane protein in a functional conformational state. 
     
     
         13 . The recombinant cell of  claim 1 , wherein the host cell is a eukaryotic cell. 
     
     
         14 . The method according to  claim 13 , wherein the host cell is a yeast selected from the group consisting of a  Pichia  strain, a Komagataella strain, a  Hansenula  strain, a  Yarrowia  strain, and a  Saccharomyces  strain. 
     
     
         15 . The method according to  claim 1 , wherein the host cell is of human origin. 
     
     
         16 . The method according to  claim 1 , wherein the host cell is an Sf9 cell. 
     
     
         17 . The method according to  claim 1 , wherein the host cell is a glycoengineered cell. 
     
     
         18 . The method according to  claim 1 , wherein the host cell is a filamentous fungi selected from the group consisting of an  Aspergillus  strain, a  Penicillium  strain, and a Hypocrea strain.