Cotranslational assembly of membrane protein/nanoparticles in cell-free systems

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dc.contributor.authorLevin, Roman
dc.contributor.authorKoeck, Zoe
dc.contributor.authorMartin, Janosch
dc.contributor.authorZangl, Rene
dc.contributor.authorGewering, Theresa
dc.contributor.authorSchueler, Leah
dc.contributor.authorMoeller, Arne
dc.contributor.authorDoetsch, Volker
dc.contributor.authorMorgner, Nina
dc.contributor.authorBernhard, Frank
dc.date.accessioned2023-02-17T11:33:34Z-
dc.date.available2023-02-17T11:33:34Z-
dc.date.issued2022
dc.identifier.issn0005-2736
dc.identifier.urihttp://osnascholar.ub.uni-osnabrueck.de/handle/unios/65379-
dc.description.abstractNanoparticles composed of amphiphilic scaffold proteins and small lipid bilayers are valuable tools for reconstitution and subsequent functional and structural characterization of membrane proteins. In combination with cell-free protein production systems, nanoparticles can be used to cotranslationally and translocon independently insert membrane proteins into tailored lipid environments. This strategy enables rapid generation of protein/ nanoparticle complexes by avoiding detergent contact of nascent membrane proteins. Frequently in use are nanoparticles assembled with engineered derivatives of either the membrane scaffold protein (MSP) or the Saposin A (SapA) scaffold. Furthermore, several strategies for the formation of membrane protein/nanoparticle complexes in cell-free reactions exist. However, it is unknown how these strategies affect functional folding, oligomeric assembly and membrane insertion efficiency of cell-free synthesized membrane proteins.We systematically studied membrane protein insertion efficiency and sample quality of cell-free synthesized proteorhodopsin (PR) which was cotranslationally inserted in MSP and SapA based nanoparticles. Three possible PR/nanoparticle formation strategies were analyzed: (i) PR integration into supplied preassembled nanoparticles, (ii) coassembly of nanoparticles from supplied scaffold proteins and lipids upon PR expression, and (iii) coexpression of scaffold proteins together with PR in presence of supplied lipids. Yield, homogeneity as well as the formation of higher PR oligomeric complexes from samples generated by the three strategies were analyzed. Conditions found optimal for PR were applied for the synthesis of a G-protein coupled receptor. The study gives a comprehensive guideline for the rapid synthesis of membrane protein/nanoparticle samples by different processes and identifies key parameters to modulate sample yield and quality.
dc.description.sponsorshipCenter for Biomolecular Magnetic Resonance; LOEWE project GLUE of the state of Hessen; DFG project BE; [1911/8-1]; We thank Birgit Schafer for technical assistance. We further thank Jens Frauenfeld for helping to establish the Salipro technology. The work was funded by the Center for Biomolecular Magnetic Resonance and by the LOEWE project GLUE of the state of Hessen. Financial support was further obtained by the DFG project BE 1911/8-1.
dc.language.isoen
dc.publisherELSEVIER
dc.relation.ispartofBIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
dc.subjectBiochemistry & Molecular Biology
dc.subjectBiophysics
dc.subjectCell-free expression
dc.subjectCOMPLEXES
dc.subjectESCHERICHIA-COLI
dc.subjectEXPRESSION
dc.subjectGPCR
dc.subjectINSERTION
dc.subjectLIPIDS
dc.subjectMembrane scaffold protein
dc.subjectNanodisc
dc.subjectNANODISCS
dc.subjectNANOPARTICLES
dc.subjectPROTEINS
dc.subjectProteorhodopsin
dc.subjectSalipro
dc.titleCotranslational assembly of membrane protein/nanoparticles in cell-free systems
dc.typejournal article
dc.identifier.doi10.1016/j.bbamem.2022.184017
dc.identifier.isiISI:000880620500007
dc.description.volume1864
dc.description.issue11
dc.identifier.eissn1879-2642
dc.publisher.placeRADARWEG 29, 1043 NX AMSTERDAM, NETHERLANDS
dcterms.isPartOf.abbreviationBiochim. Biophys. Acta-Biomembr.
local.import.remainsaffiliations : Goethe University Frankfurt; Goethe University Frankfurt; Goethe University Frankfurt; University Osnabruck
local.import.remainsweb-of-science-index : Science Citation Index Expanded (SCI-EXPANDED)
crisitem.author.deptFB 05 - Biologie/Chemie-
crisitem.author.deptidfb05-
crisitem.author.parentorgUniversität Osnabrück-
crisitem.author.netidMoAr687-
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