Functional Reconstitution of Membrane Proteins Derived From Eukaryotic Cell-Free Systems

Srujan Kumar Dondapati,Henning Lübberding,Anne Zemella,Stefan Kubick,Doreen A Wüstenhagen,Lena Thoring

doi:10.3389/fphar.2019.00917

Abstract

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.

Highlights

Cell-free synthesis of proteins (CFPS) offers several advantages over traditional approaches
HVDAC1 was synthesized in Sf21 based cell-free systems using a batch based 2h reaction
Total synthesis reaction which we mentioned as suspension (SU) is centrifuged at 16,000xg for 10 min and the pellet which we mentioned as vesicular fraction (VF) contains the protein incorporated microsomes is separated from the supernatant fraction (SN) which contains the cell-free components and other non incorporated protein as well as smaller microsomes

Summary

Introduction

Cell-free synthesis of proteins (CFPS) offers several advantages over traditional approaches. In CFPS, the protein of interest is synthesized by using the translation machinery, retained in the material formed by the lysis of cells (Carlson et al, 2012; Zemella et al, 2015). Cell-free protein synthesis (CFPS) is a fast and user-friendly technique, where the protein production process is Functional Reconstitution of Membrane Proteins completed within 2h and proteins can be analyzed immediately after the synthesis. CFPS is an open and flexible system and allows the facile modification of components related to protein translation. For efficient CFPS, supplementation of reaction components such as amino acids, RNA polymerase, salts, and energy components can be actively monitored and manipulated specific to each individual protein. Additives in CFPS substantially influence both the folding and quantity of functional protein

Methods

Results

Conclusion