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Front. Pharmacol. | doi: 10.3389/fphar.2019.00917

Functional reconstitution of membrane proteins derived from eukaryotic cell-free systems

 Srujan Kumar Dondapati1, Henning Lübberding1, Anne Zemella1, Lena Thoring1, Doreen A. Wüstenhagen1 and Stefan Kubick1*
  • 1Fraunhofer-Institut für Zelltherapie und Immunologie, Institutsteil Bioanalytik und Bioprozesse (IZI-BB), Germany

Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.

Keywords: Sf21 lysates, Microsomes, Cell-free protein synthesis (CFPS), proteoliposome, transporter, ion channel, Membrane Proteins

Received: 30 Apr 2019; Accepted: 22 Jul 2019.

Edited by:

Lei Kai, School of Life Science, Jiangsu Normal University, China

Reviewed by:

Christoph E. Hagemeyer, Monash University, Australia
Yuan Lu, Tsinghua University, China  

Copyright: © 2019 Dondapati, Lübberding, Zemella, Thoring, Wüstenhagen and Kubick. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Dr. Stefan Kubick, Fraunhofer-Institut für Zelltherapie und Immunologie, Institutsteil Bioanalytik und Bioprozesse (IZI-BB), Potsdam, 14476, Brandenburg, Germany, stefan.kubick@izi-bb.fraunhofer.de