Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles

Abstract

Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. A number of recent studies have demonstrated successful incorporation of membrane proteins into discoidal nanolipoprotein particles (NLPs) 1The abbreviations used are: NLP, nanolipoprotein particle or nanodisc; MP, membrane protein; bR, bacteriorhodopsin; bOp, bacterioOpsin; DMPC, dimyristoylphosphatidylcholine; Δ49A1, apolipoprotein A-I with amino acids 1–49 deleted; Δ55A1, apolipoprotein A-I with amino acids 1–55 deleted; AFM, atomic force microscopy; IR, infrared; TM, transmembrane; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; SEC, size exclusion chromatography. (1Bayburt T.H. Carlson J.W. Sligar S.G. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer.J. Struct. Biol. 1998; 123: 37-44Crossref PubMed Scopus (138) Google Scholar, 2Bayburt T.H. Grinkova Y.V. Sligar S.G. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.Arch. Biochem. Biophys. 2006; 450: 215-222Crossref PubMed Scopus (137) Google Scholar, 3Bayburt T.H. Sligar S.G. Self-assembly of single integral membrane proteins into soluble nanoscale phospholipid bilayers.Protein Sci. 2003; 12: 2476-2481Crossref PubMed Scopus (217) Google Scholar, 4Leitz A.J. Bayburt T.H. Barnakov A.N. Springer B.A. Sligar S.G. Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology.BioTechniques. 2006; 40 (604, 606, passim.): 601-602Crossref PubMed Scopus (181) Google Scholar). NLPs have recently been used to understand and limit the oligomerization of membrane proteins, e.g. G-protein-coupled receptors, enabling further studies critical to understanding macromolecular recognition and cellular signaling (4Leitz A.J. Bayburt T.H. Barnakov A.N. Springer B.A. Sligar S.G. Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology.BioTechniques. 2006; 40 (604, 606, passim.): 601-602Crossref PubMed Scopus (181) Google Scholar, 5Bayburt T.H. Leitz A.J. Xie G. Oprian D.D. Sligar S.G. Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.J. Biol. Chem. 2007; 282: 14875-14881Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 6Whorton M.R. Bokoch M.P. Rasmussen S.G. Huang B. Zare R.N. Kobilka B. Sunahara R.K. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7682-7687Crossref PubMed Scopus (568) Google Scholar). However, the number and type of membrane proteins studied in the context of NLPs have been limited thus far (2Bayburt T.H. Grinkova Y.V. Sligar S.G. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.Arch. Biochem. Biophys. 2006; 450: 215-222Crossref PubMed Scopus (137) Google Scholar, 4Leitz A.J. Bayburt T.H. Barnakov A.N. Springer B.A. Sligar S.G. Functional reconstitution of β2-adrenergic receptors utilizing self-assembling Nanodisc technology.BioTechniques. 2006; 40 (604, 606, passim.): 601-602Crossref PubMed Scopus (181) Google Scholar, 5Bayburt T.H. Leitz A.J. Xie G. Oprian D.D. Sligar S.G. Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.J. Biol. Chem. 2007; 282: 14875-14881Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 6Whorton M.R. Bokoch M.P. Rasmussen S.G. Huang B. Zare R.N. Kobilka B. Sunahara R.K. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7682-7687Crossref PubMed Scopus (568) Google Scholar, 7Alami M. Dalal K. Lelj-Garolla B. Sligar S.G. Duong F. Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.EMBO J. 2007; 26: 1995-2004Crossref PubMed Scopus (125) Google Scholar, 8Boldog T. Grimme S. Li M. Sligar S.G. Hazelbauer G.L. Nanodiscs separate chemoreceptor oligomeric states and reveal their signaling properties.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 11509-11514Crossref PubMed Scopus (164) Google Scholar, 9Cruz F. Edmondson D.E. Kinetic properties of recombinant MAO-A on incorporation into phospholipid nanodisks.J. Neural. Transm. 2007; 114: 699-702Crossref PubMed Scopus (21) Google Scholar). In NLPs, the apolipoproteins act as a supporting protein “scaffold” organizing the lipid into bilayers (10Chromy B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar, 11Jonas A. Reconstitution of high-density lipoproteins.Methods Enzymol. 1986; 128: 553-582Crossref PubMed Scopus (296) Google Scholar). As we have reported previously, NLPs have an average height of 5.0 ± 0.5 nm with diameters ranging from 10 to 60 nm (±3%), depending upon the apolipoprotein and lipid used to make the NLP (10Chromy B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar). These methods of in situ generation of NLPs have not been demonstrated previously in cell-free reactions, which may allow for a greater range of cofactors and additives for obtaining improved solubility and function. Such additions may also help expand the applicability of NLPs to a more diverse population of proteins. Cell-free protein expression has been used to enable protein biochemistry studies for many years (12Katzen F. Chang G. Kudlicki W. The past, present and future of cell-free protein synthesis.Trends Biotechnol. 2005; 23: Full Text Full Text PDF PubMed Scopus Google Scholar). in of membrane proteins in protein and However, a number of cell-free as to the expression of membrane proteins F. B. W. H. F. cell-free expression and of integral membrane J. Biochem. PubMed Scopus Google Scholar, A. J. W. S. F. Cell-free production of G protein-coupled receptors for functional and Struct. Biol. 2007; PubMed Scopus Google Scholar, A. B. B. F. F. of cell-free expression and as a approach for of membrane Chem. 2006; PubMed Scopus Google Scholar, M. Cell-free and of in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, T. M. T. T. A bilayer cell-free protein for of PubMed Scopus Google Scholar, A. F. B. F. cell-free expression for the of integral membrane proteins for functional and 2007; PubMed Scopus Google Scholar, S. W. Cell-free functional and of an integral membrane PubMed Scopus Google Scholar). Cell-free expression of membrane proteins can also the with of membrane protein expression may to the for lipid bilayer insertion for and function. incorporation of cell-free bacteriorhodopsin into has been reported previously two The cell-free the cell-free protein expression in the presence of S. W. Cell-free functional and of an integral membrane PubMed Scopus Google Scholar, T. E. M. Functional cell-free of a membrane in situ insertion of bacteriorhodopsin into Biol. 2007; PubMed Scopus Google Scholar). methods functional bacteriorhodopsin (bR) with limited solubility and S. W. Cell-free functional and of an integral membrane PubMed Scopus Google Scholar, T. E. M. Functional cell-free of a membrane in situ insertion of bacteriorhodopsin into Biol. 2007; PubMed Scopus Google Scholar). The study of membrane proteins in further limited in that membrane proteins into have one to the the to the membrane protein with other The co-expression of NLPs and membrane proteins cell-free methods may and of in membrane protein expression method may provide a general cell-free approach for membrane protein a range of membrane proteins for study with current The of apolipoprotein A-I or Δ49A1, was the and from the apolipoprotein The was into the and The a for The which the bR was from the S. W. Cell-free functional and of an integral membrane PubMed Scopus Google the and The was into the the and by DNA the or the In amino and in Reconstitution and as by the For co-expression a of of DNA and was to the along with and cofactor The or to the for the for for bR production was as as For membrane protein survey of DNA and of membrane DNA to the cell-free was in of The soluble was by the for by not and the of solubility was determined of to protein in the presence and of apolipoprotein all-trans-retinal was with a of or 10 The was to a of in cell-free The difference in the of or the in the not significantly the cell-free of by a of was on of force was used to from the to the cell-free a of was used to the proteins of Δ49A1 and from the cell-free The soluble was with of to the with the 10 in was used for the with of A of of in used to the protein from the in of the and into to a of was used for of NLPs (1Bayburt T.H. Carlson J.W. Sligar S.G. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer.J. Struct. Biol. 1998; 123: 37-44Crossref PubMed Scopus (138) Google Scholar, B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar). the of or Δ55A1, from Nanodisc was with in a of in The was for The NLPs by size exclusion chromatography. For of was with in a of in was to a of membrane bacteriorhodopsin W. of the membrane of and its into and Enzymol. PubMed Scopus Google was in a to the apolipoprotein and for by in for The NLPs by size exclusion chromatography. The NLPs with and membrane protein from and by a with a of 0.5 The was with protein of and that the range of the and the NLP The was with The NLP was to with was determined the protein of the cell-free soluble and with with and a along with the The was for with of NLP with and for a with protein for and a with the of a with a determined by of proteins in the NLPs an atomic force in with force and of was used for to NLP height and and from NLPs by cell-free and also for NLPs by cell-free to NLPs, and to to the height and of the NLP population in the co-expressed with bR with the with bR A of was difference spectra in the rapid to studies of in the and of PubMed Scopus Google Scholar). a was on a the and in a a with a and to the of single spectra the and used as the A the and spectra with in this to a The of this study was to cell-free methods for functional membrane protein production that and solubilize the membrane protein in a of the membrane protein are on the of membrane proteins to into lipid bilayers cell-free T. E. M. Functional cell-free of a membrane in situ insertion of bacteriorhodopsin into Biol. 2007; PubMed Scopus Google Scholar, S. W. A. S. K. of by in membrane protein Biotechnol. PubMed Scopus Google the of apolipoprotein to lipid bilayer (10Chromy B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google and the demonstrated of NLPs to solubilize the membrane proteins (2Bayburt T.H. Grinkova Y.V. Sligar S.G. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.Arch. Biochem. Biophys. 2006; 450: 215-222Crossref PubMed Scopus (137) Google Scholar, 5Bayburt T.H. Leitz A.J. Xie G. Oprian D.D. Sligar S.G. Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.J. Biol. Chem. 2007; 282: 14875-14881Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 7Alami M. Dalal K. Lelj-Garolla B. Sligar S.G. Duong F. Nanodiscs unravel the interaction between the SecYEG channel and its cytosolic partner SecA.EMBO J. 2007; 26: 1995-2004Crossref PubMed Scopus (125) Google Scholar, M.R. B. A. K. Sunahara R.K. of to monomeric in a phospholipid bilayer.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). DNA encoding the membrane protein bR from and a of apolipoprotein A-I the amino acids to cell-free along with and cofactors to produce membrane NLPs in a single as shown in bR, a has been as a protein for and other proteins as the receptor of proteins. co-expression and of the soluble was of the soluble functional The of bR to or S. W. Cell-free functional and of an integral membrane PubMed Scopus Google and cell-free protein expression of bR and Δ49A1 in the presence of in a single a functional and rapid approach was also to a of other proteins indicating the general applicability of this of membrane proteins with apolipoprotein Δ49A1 in the presence of lipid solubility of membrane proteins. A was between the membrane protein expression of the membrane protein in the presence of and membrane protein co-expressed with apolipoprotein in the presence of proteins with the number of in are: receptor and bR In all the solubility of the membrane protein increased with co-expression of from by the incorporation of into the cell-free not from of Cell-free of bR in the presence of and functional bR and the bR was in the presence of Δ49A1, of the was soluble with the Δ49A1 was co-expressed in the and as in the soluble and with and Δ49A1 methods for of cell-free bR into lipid have been reported previously S. W. Cell-free functional and of an integral membrane PubMed Scopus Google Scholar, T. E. M. Functional cell-free of a membrane in situ insertion of bacteriorhodopsin into Biol. 2007; PubMed Scopus Google Scholar). However, these two approaches a of and further by limited membrane protein because of the of In the co-expressed bR·NLPs soluble exclusion a size shift in the with NLPs or The NLPs and and A size range of was for this was the NLPs was also by and may have been to as number of NLP, bR oligomerization or of bR the NLPs, generation of NLPs with diameters by C.D. Benner Pesavento J.B. Cappuccio J.A. Walsworth Kuhn E.A. M. B.A. Segelke B.W. Coleman M.A. Bench G. Hoeprich P.D. Sulchek T.A. size of nanolipoprotein particles with and Full Text Full Text PDF PubMed Scopus Google the Functional of the soluble co-expressed was determined by adaptation The adaptation a 5-nm shift with a absorption of nm and light absorption of These that the of bR in a monomeric in the presence of NLPs a bilayer M.P. and adaptation of monomeric and bacteriorhodopsin in lipid PubMed Scopus Google Scholar, J. S. C.D. M.A. of the of the of bacteriorhodopsin in its and monomeric J. Full Text Full Text PDF PubMed Scopus Google Scholar). in with other studies that used apolipoprotein to solubilize of bR (2Bayburt T.H. Grinkova Y.V. Sligar S.G. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.Arch. Biochem. Biophys. 2006; 450: 215-222Crossref PubMed Scopus (137) Google Scholar). The of bR isolated from the soluble was from a cell-free The of approach that we in less that in a single the for of membrane protein and prior to the of protein the co-expression method to membrane proteins, a of membrane proteins with of cell-free Cell-free expression of the membrane protein and expression of the membrane protein used for The to the from and soluble by of was used to the The of solubilized membrane protein with the membrane protein was in In all co-expression with the apolipoprotein Δ49A1 was greater the expression of the membrane protein or membrane protein expression bR·NLPs from cell-free co-expression and NLPs by (10Chromy B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar, C.D. Benner Pesavento J.B. Cappuccio J.A. Walsworth Kuhn E.A. M. B.A. Segelke B.W. Coleman M.A. Bench G. Hoeprich P.D. Sulchek T.A. size of nanolipoprotein particles with and Full Text Full Text PDF PubMed Scopus Google by to NLP size and and to demonstrate the between bR with NLPs. For with cell-free bR·NLPs and NLPs also methods previously (1Bayburt T.H. Carlson J.W. Sligar S.G. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer.J. Struct. Biol. 1998; 123: 37-44Crossref PubMed Scopus (138) Google Scholar, B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar). These NLPs and with apolipoprotein A-I or Nanodisc membrane bR, and (1Bayburt T.H. Carlson J.W. Sligar S.G. Reconstitution and imaging of a membrane protein in a nanometer-size phospholipid bilayer.J. Struct. Biol. 1998; 123: 37-44Crossref PubMed Scopus (138) Google Scholar, B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google Scholar). the co-expressed and bR·NLPs showed in particle height to an NLP indicating of bR protein the NLPs NLPs by or cell-free methods of ± nm as determined by and The NLPs by of and bR or co-expression of Δ49A1 and bR as two discoidal by height The population a height of ± nm to NLPs The which was not in bR, a height of ± nm The increased height in the presence of bR in the of the NLP and with the bR the NLP lipid bilayer the increased height particles in the presence of bR also an increased bR·NLPs an average of ± nm and the an average of ± nm and ± in the presence and of bR, the in height as a for bR·NLPs from we to an of NLPs with bR incorporation of that was difference between the and height of the by cell-free methods in the presence and of bR with of and However, a in and height was between the bR·NLPs and with of and was also used to the lipid with reported in (10Chromy B.A. Arroyo E. Blanchette C.D. Bench G. Benner H. Cappuccio J.A. Coleman M.A. Henderson P.T. Hinz A.K. Kuhn E.A. Pesavento J.B. Segelke B.W. Sulchek T.A. Tarasow T. Walsworth V.L. Hoeprich P.D. Different apolipoproteins impact nanolipoprotein particle formation.J. Am. Chem. Soc. 2007; 129: 14348-14354Crossref PubMed Scopus (75) Google and The of this was in ranging from to 60 nm in and from to nm in height indicating that the of the was lipid as or membrane K. B. of from PubMed Scopus Google Scholar, E. of between and PubMed Scopus Google Scholar, A. of phospholipid bilayers from by atomic force Scopus Google Scholar, the of lipid bilayers on a study AFM, and J. 2005; Full Text Full Text PDF PubMed Scopus Google of of cell-free NLPs with and co-expressed ± ± ± ± ± ± for ± for for in a difference of type and bR·NLPs are shown in characteristic of the M the of bacteriorhodopsin as with the light → → → M → → → and M. of bacteriorhodopsin by infrared difference Biophys. 103: PubMed Scopus Google Scholar). 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The that bR to the insertion into the NLP and that bR difference further that the cell-free bR·NLPs functional spectra of bR·NLPs showed the of protein and that the bR → M of the bR in the spectra are because of of the bR·NLPs that have been in the NLP as with the of bR in membrane in bacteriorhodopsin the by time-resolved infrared J. Full Text Full Text PDF PubMed Scopus Google Scholar, infrared absorption of PubMed Scopus Google Scholar). Importantly the was to the for the of Cell-free production of proteins has as a to with protein expression and in of of soluble proteins T. M. T. T. A bilayer cell-free protein for of PubMed Scopus Google Scholar, A. F. B. F. cell-free expression for the of integral membrane proteins for functional and 2007; PubMed Scopus Google Scholar, T. T. S. protein the cell-free Google Scholar, B.W. J. Coleman M.A. B. Struct. 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F. of cell-free expression and as a approach for of membrane Chem. 2006; PubMed Scopus Google S. of the of 2006; 12: PubMed Scopus Google Scholar). cell-free NLPs also enable for for membrane protein expression a of additives that may protein production and additives J. of protein between in and in PubMed Scopus Google K. W. F. of for the soluble expression of and integral membrane proteins by a cell-free expression J. 2005; PubMed Scopus Google expression and of of protein cell-free protein PubMed Scopus Google and K. W. F. of for the soluble expression of and integral membrane proteins by a cell-free expression J. 2005; PubMed Scopus Google Scholar). As previously, a limited number of G-protein-coupled receptors and proteins as bR have been into NLPs lipids, (2Bayburt T.H. Grinkova Y.V. Sligar S.G. Assembly of single bacteriorhodopsin trimers in bilayer nanodiscs.Arch. Biochem. Biophys. 2006; 450: 215-222Crossref PubMed Scopus (137) Google T.H. Leitz A.J. Xie G. Oprian D.D. Sligar S.G. Transducin activation by nanoscale lipid bilayers containing one and two rhodopsins.J. Biol. Chem. 2007; 282: 14875-14881Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar, 9Cruz F. Edmondson D.E. Kinetic properties of recombinant MAO-A on incorporation into phospholipid nanodisks.J. Neural. Transm. 2007; 114: 699-702Crossref PubMed Scopus (21) Google or a of and M.R. Bokoch M.P. Rasmussen S.G. Huang B. Zare R.N. Kobilka B. Sunahara R.K. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein.Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 7682-7687Crossref PubMed Scopus (568) Google Scholar). cell-free by single addition of and other to protein function. The from this single of integral membrane proteins expand the of for cell-free protein expression and to production of soluble membrane proteins by a nanolipoprotein with