title: Express incorporation of membrane proteins from various human cell types into phospholipid bilayer nanodiscs.
abstract: Analysis of membrane proteins is still inadequately represented in diagnostics despite their importance as drug targets and biomarkers. One main reason is the difficult handling caused by their insolubility in aqueous buffer solutions. The nanodisc technology was developed to circumvent this challenge and enables analysis of membrane proteins with standard research methods. However, existing nanodisc generation protocols rely on time-consuming membrane isolation and protein purification from overexpression systems. In the present study, we present a novel, simplified procedure for the rapid generation of nanodiscs directly from intact cells. Workflow and duration of the nanodisc preparation were shortened without reducing the reconstitution efficiency, and all the steps were modified for the use of only standard laboratory equipment. This protocol was successfully applied to various human cell types, such as cultivated human embryonic kidney 293 (HEK-293) cells, as well as freshly isolated human red blood cells and platelets. In addition, the reconstitution of membrane proteins from cell organelles was achieved. The use of endogenous lipids ensures a native-like environment, which promotes native protein (re)folding. Nanodisc generation was verified by size exclusion chromatography and EM, whereas incorporation of different membrane proteins was demonstrated by Western blot analysis. Our protocol enabled the rapid incorporation of endogenous membrane proteins from human cells into nanodiscs, which can be applied to analytical approaches.
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Mak
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany.
authors.forename: Ruoyu
authors.initials: R
authors.lastname: Sun
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Schmalenberg
authors.affiliation: Department Chemistry, Centre for Integrated Protein Science Munich (CiPSM), Technical University of Munich, Lichtenbergstrasse 4, Munich 85748, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Peters
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technical University of Munich, Ismaninger Strasse 22, Munich 81675, Germany p.luppa@tum.de.
authors.forename: Peter B
authors.initials: PB
authors.lastname: Luppa
mesh_terms: D001792:Blood Platelets / Q000378:metabolism* / Q000648:ultrastructure; D002850:Chromatography, Gel; D004912:Erythrocytes / Q000378:metabolism* / Q000648:ultrastructure; D057809:HEK293 Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D049329:Nanostructures / Q000737:chemistry / Q000648:ultrastructure; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D058849:Protein Refolding
chemical_list: D008051:Lipid Bilayers; D008563:Membrane Lipids; D008565:Membrane Proteins; D010743:Phospholipids
keywords: HEK-293 cells; erythrocytes; express protocol; nanodiscs; platelets