Qualifying a eukaryotic cell-free system for fluorescence based GPCR analyses

Membrane proteins are key elements in cell-mediated processes. In particular, G protein-coupled receptors (GPCRs) have attracted increasing interest since they affect cellular signaling. Furthermore, mutations in GPCRs can cause acquired and inheritable diseases. Up to date, there still exist a number of GPCRs that has not been structurally and functionally analyzed due to difficulties in cell-based membrane protein production. A promising approach for membrane protein synthesis and analysis has emerged during the last years and is known as cell-free protein synthesis (CFPS). Here, we describe a simply portable method to synthesize GPCRs and analyze their ligand-binding properties without the requirement of additional supplements such as liposomes or nanodiscs. This method is based on eukaryotic cell lysates containing translocationally active endogenous endoplasmic reticulum-derived microsomes where the insertion of GPCRs into biologically active membranes is supported. In this study we present CFPS in combination with fast fluorescence-based screening methods to determine the localization, orientation and ligand-binding properties of the endothelin B (ET-B) receptor upon expression in an insect-based cell-free system. To determine the functionality of the cell-free synthesized ET-B receptor, we analyzed the binding of its ligand endothelin-1 (ET-1) in a qualitative fluorescence-based assay and in a quantitative radioligand binding assay

Membrane protein synthesis in cell-free systems: From bio-mimetic systems to bio-membranes

AbstractWhen taking up the gauntlet of studying membrane protein functionality, scientists are provided with a plethora of advantages, which can be exploited for the synthesis of these difficult-to-express proteins by utilizing cell-free protein synthesis systems. Due to their hydrophobicity, membrane proteins have exceptional demands regarding their environment to ensure correct functionality. Thus, the challenge is to find the appropriate hydrophobic support that facilitates proper membrane protein folding. So far, various modes of membrane protein synthesis have been presented. Here, we summarize current state-of-the-art methodologies of membrane protein synthesis in biomimetic-supported systems. The correct folding and functionality of membrane proteins depend in many cases on their integration into a lipid bilayer and subsequent posttranslational modification. We highlight cell-free systems utilizing the advantages of biological membranes

Membranproteinsynthese: Zellfrei geht's schneller!: Zellfreie Proteinproduktion

Difficult to express membrane proteins represent an increasing amount of therapeutic molecules. Considerable optimization is often required for downstream applications such as assay development and functional characterization. Cell-free systems emerged as powerful tools for the synthesis of structurally and functionally divergent membrane proteins. Vesicle-based eukaryotic cell-free systems enable co-translational protein translocation and posttranslational modifications. Hence, these systems provide a multitude of options for membrane protein studies

The <em>Salmonella</em> Deubiquitinase SseL Inhibits Selective Autophagy of Cytosolic Aggregates

<div><p>Cell stress and infection promote the formation of ubiquitinated aggregates in both non-immune and immune cells. These structures are recognised by the autophagy receptor p62/sequestosome 1 and are substrates for selective autophagy. The intracellular growth of <em>Salmonella enterica</em> occurs in a membranous compartment, the <em>Salmonella</em>-containing vacuole (SCV), and is dependent on effectors translocated to the host cytoplasm by the <em>Salmonella</em> pathogenicity island-2 (SPI-2) encoded type III secretion system (T3SS). Here, we show that bacterial replication is accompanied by the formation of ubiquitinated structures in infected cells. Analysis of bacterial strains carrying mutations in genes encoding SPI-2 T3SS effectors revealed that in epithelial cells, formation of these ubiquitinated structures is dependent on SPI-2 T3SS effector translocation, but is counteracted by the SPI-2 T3SS deubiquitinase SseL. In macrophages, both SPI-2 T3SS-dependent aggregates and aggresome-like induced structures (ALIS) are deubiquitinated by SseL. In the absence of SseL activity, ubiquitinated structures are recognized by the autophagy receptor p62, which recruits LC3 and targets them for autophagic degradation. We found that SseL activity lowers autophagic flux and favours intracellular <em>Salmonella</em> replication. Our data therefore show that there is a host selective autophagy response to intracellular <em>Salmonella</em> infection, which is counteracted by the deubiquitinase SseL.</p> </div

Time course analysis of the cell-free synthesized type-I transmembrane protein Mel-Hb-EGF-eYFP.

<p>Batch and CECF reactions were carried out in the presence of caspase inhibitor and ¹⁴C-leucine and in the absence of DTT. Protein yields in batch (A) and CECF reactions (B) were determined in the translation mixture and the vesicular fraction by liquid scintillation counting. Standard deviations were calculated from triplicate analysis (n = 3). C) Qualitative analysis of Mel-Hb-EGF-eYFP in the translation mixture by SDS-PAGE and autoradiography. D) Analysis of Mel-Hb-EGF-eYFP in the vesicular fraction. Cell-free synthesized Mel-Hb-EGF-eYFP shows a migration pattern corresponding to its expected molecular mass (calculated molecular mass  = 51 kDa). NTC  =  No template control; translation reaction without addition of a DNA template.</p

Time course of ¹⁴C-leucine labeled eYFP synthesized in batch and CECF mode.

<p>Cell-free reactions using insect lysate were carried out in the presence (+) and absence (−) of insect vesicles (V) and caspase inhibitor (CI). Translation mixtures were analyzed by SDS-PAGE and autoradiography. Cell-free synthesized eYFP shows a migration pattern corresponding to its expected molecular mass (calculated molecular mass  = 29 kDa).</p

Influence of caspase inhibitor (CI) on the expression yield of representative model proteins.

<p>The membrane proteins Mel-Hb-EGF-eYFP (51 kDa), bacteriorhodopsin (27 kDa) and endothelin-B receptor (49 kDa), the glycoproteins Mel-EPO (21 kDa, protein in its non-glycosylated form) and Mel-vtPA (41 kDa, protein in its non-glycosylated form) as well as the protein eYFP (29 kDa) were synthesized in batch (B) and CECF (C) reactions in the presence (+) and absence (−) of CI using insect lysate. Reactions were carried out for 48 h in the absence of the reducing agent DTT and in presence of ¹⁴C-leucine. A) Diagram showing total protein yields which were determined by incorporation of ¹⁴C-leucine and liquid scintillation counting. Standard deviations were calculated from triplicate analysis (n = 3). B) Fluorescence imaging of eYFP and Mel-Hb-EGF-eYFP using a phosphorimager system. NTC  =  No template control; translation reaction without addition of a DNA template. C) Qualitative analysis of cell-free synthesized proteins by SDS-PAGE and autoradiography. Addition of CI significantly increased the protein yield of all target proteins analyzed in this study.</p

Analysis of Mel-vtPA expression and activity.

<p>Cell-free reactions were performed for 48 h in batch and CECF mode in absence of DTT, but in presence of caspase inhibitor and ¹⁴C-leucine using three differently composed reaction mixtures. (i) lysate without GSH and GSSG; (ii) lysate supplemented with GSH and GSSG and (iii) buffer and lysate supplemented with GSH and GSSG. A) Diagram showing total yields of ¹⁴C-leucine labeled Mel-vtPA. B) Fibrin-agarose-plate showing lytic zones created by biologically active Mel-vtPA. All samples were diluted to 0.5 µg/ml. Activity of Mel-vtPA is completely abolished after addition of 5 mM DTT. C) Activity of Mel-vtPA analyzed by fibrin-agarose-plate assay. D) Activity of the positive control ( =  purified full length tPA, 0.5 µg/ml, Anaspec) analyzed by fibrin-agarose-plate assay. NTC  =  No template control; translation reaction without addition of a DNA template. Standard deviations were calculated from triplicate analysis (n = 3).</p

Time course for the cell-free expression of eYFP.

<p>Reactions were carried out using an <i>in vitro</i> translation system based on insect lysates in batch and CECF mode in the presence (+) and absence (−) of insect vesicles (V) and caspase inhibitor (CI). A) Fluorescence imaging of eYFP using a phosphorimager system. B) Relative fluorescence intensity of eYFP in batch reactions. C) Relative fluorescence intensity of eYFP in CECF reactions. The percentage calculation of the fluorescence intensity is depicted by the fluorescence intensity of eYFP measured after 2 h of incubation set as 100% (batch, + V, - CI). For each data point fluorescence intensity of eYFP is presented as mean value of duplicate analysis, with the error bar indicating value 1 and value 2 of the duplicate. NTC  =  No template control; translation reaction without addition of a DNA template.</p