Nanoscopic leg irons: harvesting of polymer-stabilized membrane proteins with antibody-functionalized silica nanoparticles

Abstract

Silica-based nanoparticles (SiNPs) are presented to harvest complex membrane proteins, which have been embedded into unilammelar polymersomes via in vitro membrane assisted protein synthesis (iMAP). Size-optimized SiNPs have been surface-modified with polymer-targeting antibodies, which are employed to harvest the protein-containing polymersomes. The polymersomes mimic the cellular membrane. They are chemically defined and preserve their structural-functional integrity as virtually any membrane protein species can be synthesized into such architecture via the ribosomal context of a cellular lysate. The SiNPs resemble 'heavy leg irons' catching the polymersomes in order to enable gravity-based generic purification and concentration of such proteopolymersomes from the crude mixture of cellular lysates.

Synthesis and Functional Reconstitution of Light‐Harvesting Complex II into Polymeric Membrane Architectures

“…To minimize this effect, we loaded the polymersomes with 0.4 m trehalose,which is an established method for increasing stability. [19] Integration of LHCII into the polymersomes was confirmed by using purification with ac ombination of immunoprecipitation [20] and sodium carbonate extraction as described by Fujiki et al, [21] with minor adaptations and modifications (see the Supporting Information). According to this method, ahighly ionic buffer disrupts simple adsorption of proteins to the polymersome membrane surface and thus extracts partially embedded proteins.H owever proteins that are properly integrated into the membrane plane would be resistant to extraction.…”

“…According to this method, ahighly ionic buffer disrupts simple adsorption of proteins to the polymersome membrane surface and thus extracts partially embedded proteins.H owever proteins that are properly integrated into the membrane plane would be resistant to extraction. Our polymersomes were purified by using an anti-polyethylene glycol (a-PEG) antibody immobilized onto silica nanoparticles (SiNP) as described by our group, [20] which allows integrated LHCII to be separated from components of the cell lysate.T his is possible because the a-PEG antibody is able to bind to the poly(ethylene oxide) part of the polymer.F igure 3s hows an example result, where the sodium carbonate treated samples indicate that iMAPSgenerate LHCII is fully incorporated into the polymeric membrane.T he specific signal indicates the presence of LHCII as an integral membrane protein. Loss of signal strength is caused by the additional carbonate treatment.…”

“…[20] In case of tethered polymer bilayers,g old-coated( 50 nm) glass surfaces were used and SPR/SPFS experiments were performedw ithin as elf-built set-up.F luorescence measurements were made using aluminescence spectrometer(LS 55, PerkinElmer Instr.). …”

Funktionelle Synthese des Lichtsammelkomplexes II in Polymermembran‐Architekturen

“…Polymersomen mit integrierten LHCII-Komplexen, so genannten Proteopolymersomen (PPs), wurden, wie von Nallani et al beschrieben, gereinigt. [9] Die resultierenden PPs wurden auf zwei [19] Die tatsächliche Integration von LHCII in die Polymersomen wurde durch eine Kombination von Immunpräzipita-tion [20] und Natriumcarbonatextraktion bestätigt, [21] [20] beschrieben, wobei vollständig integrierter LHCII vom verwendeten Zelllysat abgetrennt wurde.D ies ist mçg-lich, da die a-PEG-Antikçrper in der Lage sind, an den Polyethylenoxidanteil des Polymers zu binden. In Abbildung 3 ist ein exemplarisches Ergebnis dieser Aufreinigung dargestellt, wobei die Natriumcarbonat-behandelte Probe zeigt, dass der mittels iMAPS synthetisierte LHCII vollständig in die Polymermembran integriert ist.…”

“…In beiden Präparationsmethoden zeigte sich die Te ndenz zur Verformung der PPs.Zur Minimierung dieses Effekts wurden die Polymersomen bereits bei der Herstellung mit 0.4 m Tr ehalose gefüllt, einer etablierten Methode zur Erhçhung der Stabilität solcher Strukturen. [19] Die tatsächliche Integration von LHCII in die Polymersomen wurde durch eine Kombination von Immunpräzipitation [20] und Natriumcarbonatextraktion bestätigt, [21] wie von Fujiki et al beschrieben. Dabei wurden nur geringfügige Anpassungen und Modifikationen in der Vorgehensweise vorgenommen (siehe Hintergrundinformationen).…”