19 results on '"Davide Proverbio"'
Search Results
2. Differential recognition of Haemophilus influenzae whole bacterial cells and isolated lipooligosaccharides by galactose-specific lectins
- Author
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Teodor Aastrup, Joseph W. St. Geme, Begoña Euba, Dolores Solís, Ioanna Kalograiaki, Junkal Garmendia, Davide Proverbio, F. Javier Cañada, María del Carmen Fernández-Alonso, Ministerio de Economía y Competitividad (España), Diputación Foral de Navarra, Instituto de Salud Carlos III, European Commission, Kalograiaki, Ioanna, Euba, Begoña, Proverbio, Davide, Aastrup, Teodor, Garmendia, Juncal, Cañada, F. Javier, Solís, Dolores, Kalograiaki, Ioanna [0000-0001-7950-2334], Euba, Begoña [0000-0001-5620-596X], Proverbio, Davide [0000-0001-6660-9298], Aastrup, Teodor [0000-0002-9535-528X], Garmendia, Juncal [0000-0002-7440-2737], Cañada, F. Javier [0000-0003-4462-1469], and Solís, Dolores [0000-0002-8148-1875]
- Subjects
Lipopolysaccharides ,0301 basic medicine ,AB-type lectin ,Identification ,Sialic-acid ,lcsh:Medicine ,Lipopolysaccharide ,Expression ,Molecular Dynamics Simulation ,medicine.disease_cause ,Article ,Epitope ,Ligand-binding characteristics ,Haemophilus influenzae ,03 medical and health sciences ,chemistry.chemical_compound ,Antigen ,medicine ,Protein glycosylation ,lcsh:Science ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Ricinus-communis agglutinin ,Antigens, Bacterial ,Multidisciplinary ,biology ,lcsh:R ,Carbohydrate interactions ,Galactose ,Lectin ,Microarray Analysis ,biology.organism_classification ,Bacterial Typing Techniques ,3. Good health ,Sialic acid ,030104 developmental biology ,chemistry ,Biochemistry ,biology.protein ,Biological Assay ,lcsh:Q ,Plant Lectins ,Glycoprotein ,Mistletoe-lectin ,Bacteria - Abstract
12 p.-4 fig.-4 tab., Bacterial surfaces are decorated with carbohydrate structures that may serve as ligands for host receptors. Based on their ability to recognize specific sugar epitopes, plant lectins are extensively used for bacteria typing. We previously observed that the galactose-specific agglutinins from Ricinus communis (RCA) and Viscum album (VAA) exhibited differential binding to nontypeable Haemophilus influenzae (NTHi) clinical isolates, their binding being distinctly affected by truncation of the lipooligosaccharide (LOS). Here, we examined their binding to the structurally similar LOS molecules isolated from strains NTHi375 and RdKW20, using microarray binding assays, saturation transfer difference NMR, and molecular dynamics simulations. RCA bound the LOSRdKW20 glycoform displaying terminal Gal beta(1,4) Glc beta, whereas VAA recognized the Gala(1,4) Gal beta(1,4) Glc beta epitope in LOSNTHi375 but not in LOSRdKW20, unveiling a different presentation. Binding assays to whole bacterial cells were consistent with LOSNTHi375 serving as ligand for VAA, and also suggested recognition of the glycoprotein HMW1. Regarding RCA, comparable binding to NTHi375 and RdKW20 cells was observed. Interestingly, an increase in LOSNTHi375 abundance or expression of HMW1 in RdKW20 impaired RCA binding. Overall, the results revealed that, besides the LOS, other carbohydrate structures on the bacterial surface serve as lectin ligands, and highlighted the impact of the specific display of cell surface components on lectin binding., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness(Grants BFU2015-70052-R, CTQ2015-64597-C2-2-P and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 03/2016), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO(Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (Grant PITN-GA-2013-608180).
- Published
- 2018
3. Combined Bacteria Microarray and Quartz Crystal Microbalance Approach for Exploring Glycosignatures of Nontypeable Haemophilus influenzae and Recognition by Host Lectins
- Author
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Teodor Aastrup, Ioanna Kalograiaki, Begoña Euba, Davide Proverbio, Juncal Garmendia, Dolores Solís, María Asunción Campanero-Rhodes, Ministerio de Economía y Competitividad (España), Centro de Investigación Biomédica en Red Enfermedades Respiratorias (España), and European Commission
- Subjects
0301 basic medicine ,Microarray ,biology ,Chemistry ,Microarray analysis techniques ,Quartz Crystal Microbalance Techniques ,Quartz crystal microbalance ,Microarray Analysis ,biology.organism_classification ,medicine.disease_cause ,Haemophilus influenzae ,Epitope ,3. Good health ,Analytical Chemistry ,Microbiology ,03 medical and health sciences ,030104 developmental biology ,Polysaccharides ,Lectins ,medicine ,DNA microarray ,Bacteria - Abstract
Recognition of bacterial surface epitopes by host receptors plays an important role in the infectious process and is intimately associated with bacterial virulence. Delineation of bacteria-host interactions commonly relies on the detection of binding events between purified bacteria- and host-target molecules. In this work, we describe a combined microarray and quartz crystal microbalance (QCM) approach for the analysis of carbohydrate-mediated interactions directly on the bacterial surface, thus preserving the native environment of the bacterial targets. Nontypeable Haemophilus influenzae (NTHi) was selected as a model pathogenic species not displaying a polysaccharide capsule or O-antigen-containing lipopolysaccharide, a trait commonly found in several important respiratory pathogens. Here, we demonstrate the usefulness of NTHi microarrays for exploring the presence of carbohydrate structures on the bacterial surface. Furthermore, the microarray approach is shown to be efficient for detecting strain-selective binding of three innate immune lectins, namely, surfactant protein D, human galectin-8, and Siglec-14, to different NTHi clinical isolates. In parallel, QCM bacteria-chips were developed for the analysis of lectin-binding kinetics and affinity. This novel QCM approach involves capture of NTHi on lectin-derivatized chips followed by formaldehyde fixation, rendering the bacteria an integrated part of the sensor chip, and subsequent binding assays with label-free lectins. The binding parameters obtained for selected NTHi-lectin pairs provide further insights into the interactions occurring at the bacterial surface., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012-317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were funded by Marie Curie contracts from the European Commission.
- Published
- 2016
4. Label-Free Cell-Based Assay for Characterization of Biomolecules and Receptors
- Author
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Diluka, Peiris, Teodor, Aastrup, Samuel, Altun, Camilla, Käck, Maria, Gianneli, Davide, Proverbio, and Lars M, Jørgensen
- Subjects
Kinetics ,Cell Membrane ,Quartz Crystal Microbalance Techniques ,Humans ,Proteins ,Biosensing Techniques ,Protein Binding - Abstract
We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.
- Published
- 2018
5. Bacterial Surface Glycans: Microarray and QCM Strategies for Glycophenotyping and Exploration of Recognition by Host Receptors
- Author
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Juncal Garmendia, Ioanna Kalograiaki, María Asunción Campanero-Rhodes, Davide Proverbio, Dolores Solís, Begoña Euba, Teodor Aastrup, Ministerio de Economía y Competitividad (España), Diputación Foral de Navarra, Centro de Investigación Biomédica en Red Enfermedades Respiratorias (España), Instituto de Salud Carlos III, and European Commission
- Subjects
0301 basic medicine ,Glycan ,Innate immune system ,030102 biochemistry & molecular biology ,Microarray ,biology ,Bacterial Glycan ,Computational biology ,Epitope ,3. Good health ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,biology.protein ,Avidity ,DNA microarray ,Receptor - Abstract
Bacterial surfaces are decorated with a diversity of carbohydrate structures that play important roles in the bacteria–host relationships. They may offer protection against host defense mechanisms, elicit strong antigenic responses, or serve as ligands for host receptors, including lectins of the innate immune system. Binding by these lectins may trigger defense responses or, alternatively, promote attachment, thereby enhancing infection. The outcome will depend on the particular bacterial surface landscape, which may substantially differ among species and strains. In this chapter, we describe two novel methods for exploring interactions directly on the bacterial surface, based on the generation of bacterial microarrays and quartz crystal microbalance (QCM) sensor chips. Bacterial microarrays enable profiling of accessible carbohydrate structures and screening of their recognition by host receptors, also providing information on binding avidity, while the QCM approach allows determination of binding affinity and kinetics. In both cases, the chief element is the use of entire bacterial cells, so that recognition of the bacterial glycan epitopes is explored in their natural environment., We gratefully acknowledge financial support from the Spanish Ministry of Economy and Competitiveness (Grants BFU2012-36825, BFU2015-70052-R, SAF2012-31166, and SAF2015-66520-R), the Department of Health of the Navarra Government (ref. 359/2012), the CIBER of Respiratory Diseases (CIBERES), an initiative from the Spanish Institute of Health Carlos III (ISCIII), and the Marie Curie Initial Training Networks DYNANO (Grant PITN-GA-2011-289033), GLYCOPHARM (Grant PITN-GA-2012- 317297), and WntsApp (GA-No. 608180, FP7-PEOPLE-2013). I.K. and D.P. were beneficiaries of Marie Curie contracts from the European Commission.
- Published
- 2018
6. Label-Free Cell-Based Assay for Characterization of Biomolecules and Receptors
- Author
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Davide Proverbio, Lars M. Jørgensen, Teodor Aastrup, Maria Gianneli, Camilla Käck, Samuel Altun, and Diluka Peiris
- Subjects
0301 basic medicine ,chemistry.chemical_classification ,Analyte ,Chemistry ,Biomolecule ,Cell ,Context (language use) ,Quartz crystal microbalance ,Cell membrane ,03 medical and health sciences ,030104 developmental biology ,medicine.anatomical_structure ,medicine ,Biophysics ,Receptor ,Biosensor - Abstract
We present a method to study the interaction between biomolecules and receptors present on the cell surface. This enables studies of molecular interactions in a natural biological context. As the analyte interacts with the receptors still intact on the cell surface, the experimental data provides complete dynamics and complexity of the interaction, thereby generating highly informative data. Attana's cell-based biosensor platform can be used to obtain this information from a diverse range of interactions as described in these protocols, which detail how to grow or capture cells on a surface, how to stabilize and visualize the cells on the surface, and how to set up assays to measure detailed interaction kinetics directly on the cell surface.
- Published
- 2018
7. Cell-free expression of G-protein-coupled receptors
- Author
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Erika, Orbán, Davide, Proverbio, Stefan, Haberstock, Volker, Dötsch, and Frank, Bernhard
- Subjects
Cell Extracts ,Protein Folding ,Cell-Free System ,Solubility ,Detergents ,Liposomes ,Escherichia coli ,Nanostructures ,Protein Binding ,Receptors, G-Protein-Coupled - Abstract
Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.
- Published
- 2014
8. Co-translational stabilization of insoluble proteins in cell-free expression systems
- Author
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Lei, Kai, Erika, Orbán, Erik, Henrich, Davide, Proverbio, Volker, Dötsch, and Frank, Bernhard
- Subjects
Protein Folding ,Cell-Free System ,Protein Stability ,Protein Biosynthesis ,Animals ,Humans ,Membrane Proteins ,Recombinant Proteins - Abstract
Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.
- Published
- 2014
9. Cell-Free Expression of G-Protein-Coupled Receptors
- Author
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Davide Proverbio, Frank Bernhard, Erika Orbán, Stefan Haberstock, and Volker Dötsch
- Subjects
Membrane protein ,Chemistry ,Biophysics ,Protein folding ,Class C GPCR ,Immune receptor ,Plasma protein binding ,Signal transduction ,Rhodopsin-like receptors ,G protein-coupled receptor - Abstract
Cell-free expression has emerged as a new standard for the production of membrane proteins. The reduction of expression complexity in cell-free systems eliminates central bottlenecks and allows the reliable and efficient synthesis of many different types of membrane proteins. Furthermore, the open accessibility of cell-free reactions enables the co-translational solubilization of cell-free expressed membrane proteins in a large variety of supplied additives. Hydrophobic environments can therefore be adjusted according to the requirements of individual membrane protein targets. We present different approaches for the preparative scale cell-free production of G-protein-coupled receptors using the extracts of Escherichia coli cells. We exemplify expression conditions implementing detergents, nanodiscs, or liposomes. The generated protein samples could be directly used for further functional characterization.
- Published
- 2014
10. Co-translational Stabilization of Insoluble Proteins in Cell-Free Expression Systems
- Author
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Davide Proverbio, Erik Henrich, Frank Bernhard, Erika Orbán, Lei Kai, and Volker Dötsch
- Subjects
Sample quality ,Protein stability ,Biochemistry ,Membrane protein ,law ,Chemistry ,Recombinant DNA ,Protein precipitation ,Protein folding ,Chemical chaperone ,law.invention - Abstract
Precipitation, aggregation, and inclusion body (IB) formation are frequently observed problems upon overexpression of recombinant proteins. The open accessibility of cell-free reactions allows addressing such critical steps by the addition of protein stabilizers such as chemical chaperones or detergents directly into the expression reactions. This approach could therefore reduce or even prevent initial protein precipitation already in the translation environment. The strategy might be considered to generally improve protein sample quality and to rescue proteins that are difficult to refold from IBs or from aggregated precipitates. We describe a protocol for the co-translational stabilization of difficult proteins by their expression in the presence of supplements such as alcohols, poly-ions, or detergents. We compile potentially useful compounds together with their recommended stock and working concentrations. Examples of screening experiments in order to systematically identify compounds or compound mixtures that stabilize particular proteins of interest are given. The method can primarily be considered for the production of unstable soluble proteins or of membrane proteins containing larger soluble domains.
- Published
- 2014
11. High-level cell-free production of membrane proteins with nanodiscs
- Author
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Christian, Roos, Lei, Kai, Stefan, Haberstock, Davide, Proverbio, Umesh, Ghoshdastider, Yi, Ma, Slawomir, Filipek, Xiaoning, Wang, Volker, Dötsch, and Frank, Bernhard
- Subjects
Models, Molecular ,Base Sequence ,Cell-Free System ,Solubility ,Protein Conformation ,Protein Biosynthesis ,Fermentation ,Lipid Bilayers ,Escherichia coli ,Membrane Proteins ,Nanotechnology ,DNA-Directed RNA Polymerases ,Plasmids - Abstract
This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.
- Published
- 2014
12. High-Level Cell-Free Production of Membrane Proteins with Nanodiscs
- Author
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Lei Kai, Umesh Ghoshdastider, Yi Ma, Frank Bernhard, Slawomir Filipek, Davide Proverbio, Stefan Haberstock, Xiaoning Wang, Volker Dötsch, and Christian Roos
- Subjects
Folding (chemistry) ,Membrane protein ,Chemistry ,Biophysics ,Cell free ,Target protein ,Lipid bilayer - Abstract
This chapter addresses two major bottlenecks in cell-free membrane protein production. Firstly, we describe the optimization of expression templates for obtaining membrane proteins in preparative scales. We present details for a newly established tag variation screen providing high success rates in improving expression efficiencies while having only minimal impacts on the target protein structure. Secondly, we present protocols for the efficient co-translational insertion of membrane proteins into defined lipid bilayers. We describe the production of nanodiscs and their implementation into cell-free expression reactions for the co-translational reconstitution of membrane proteins. In addition we give guidelines for the loading of nanodiscs with different lipids in order to systematically analyze effects of lipids on the translocation, functional folding, and stability of cell-free expressed membrane proteins.
- Published
- 2013
13. Functional properties of cell-free expressed human endothelin A and endothelin B receptors in artificial membrane environments
- Author
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Frank Bernhard, Davide Proverbio, Michael Beyermann, Erika Orbán, Volker Dötsch, and Christian Roos
- Subjects
Protein Folding ,Detergents ,Synthetic membrane ,Biophysics ,Gene Expression ,Biochemistry ,Lipid screening ,chemistry.chemical_compound ,Cell-free expression ,Humans ,Surface plasmon resonance ,Receptor ,POPC ,Nanodisc ,G protein-coupled receptor ,Vasoactive peptide ,biology ,Cell-Free System ,Endothelin-1 ,Cell Biology ,Receptor, Endothelin A ,Receptor, Endothelin B ,Nanostructures ,Kinetics ,chemistry ,Rhodopsin ,G-protein coupled receptor ,Liposomes ,biology.protein ,Human endothelin system ,Endothelin receptor ,Protein Binding - Abstract
The human endothelin receptors are members of the rhodopsin class A of G-protein coupled receptors and key modulators of blood pressure regulation. Their functional in vitro characterization has widely been limited by the availability of high quality samples. We have optimized cell-free expression protocols for the human endothelin A and endothelin B receptors by implementing co-translational association approaches of the synthesized proteins with supplied liposomes or nanodiscs. Efficiency of membrane association and ligand binding properties of the receptors have systematically been studied in correlation to different membrane environments and lipid types. Ligand binding was analyzed by a number of complementary assays including radioassays, surface plasmon resonance and fluorescence measurements. High affinity binding of the peptide ligand ET-1 to both endothelin receptors could be obtained with several conditions and the highest Bmax values were measured in association with nanodiscs. We could further obtain the characteristic differential binding pattern of the two endothelin receptors with a panel of selected agonists and antagonists. Two intrinsic properties of the functionally folded endothelin B receptor, the proteolytic processing based on conformational recognition as well as the formation of SDS-resistant complexes with the peptide ligand ET-1, were observed with samples obtained from several cell-free expression conditions. High affinity and specific binding of ligands could furthermore be obtained with non-purified receptor samples in crude cell-free reaction mixtures, thus providing new perspectives for fast in vitro screening applications.
- Published
- 2013
14. Co-translational association of cell-free expressed membrane proteins with supplied lipid bilayers
- Author
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Davide Proverbio, Volker Dötsch, Christian Roos, Umesh Ghoshdastider, Lei Kai, Frank Bernhard, and Slawomir Filipek
- Subjects
Peripheral membrane protein ,Detergents ,Lipid Bilayers ,Membrane Proteins ,Biological membrane ,Cell Biology ,Biology ,Membrane transport ,Protein Transport ,Biochemistry ,Membrane protein ,Protein Biosynthesis ,Liposomes ,Membrane fluidity ,Nanotechnology ,Protein–lipid interaction ,Lipid bilayer ,Molecular Biology ,Integral membrane protein - Abstract
Routine strategies for the cell-free production of membrane proteins in the presence of detergent micelles and for their efficient co-translational solubilization have been developed. Alternatively, the expression in the presence of rationally designed lipid bilayers becomes interesting in particular for biochemical studies. The synthesized membrane proteins would be directed into a more native-like environment and cell-free expression of transporters, channels or other membrane proteins in the presence of supplied artificial membranes could allow their subsequent functional analysis without any exposure to detergents. In addition, lipid-dependent effects on activity and stability of membrane proteins could systematically be studied. However, in contrast to the generally efficient detergent solubilization, the successful stabilization of membrane proteins with artificial membranes appears to be more difficult. A number of strategies have therefore been explored in order to optimize the co-translational association of membrane proteins with different forms of supplied lipid bilayers including liposomes, bicelles, microsomes or nanodiscs. In this review, we have compiled the current state-of-the-art of this technology and we summarize parameters which have been indicated as important for the co-translational association of cell-free synthesized membrane proteins with supplied membranes.
- Published
- 2012
15. Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase
- Author
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Daniela Münch, Daniel J. Müller, Volker Dötsch, Hans-Georg Sahl, Michael Zocher, Tanja Schneider, Yi Ma, Erik Henrich, Christian Roos, Josef Wachtveitl, Davide Proverbio, Frank Bernhard, and Frank Scholz
- Subjects
Rhodopsin ,Lipid Bilayers ,Biophysics ,Transferases (Other Substituted Phosphate Groups) ,medicine.disease_cause ,Biochemistry ,Antiporters ,Lipid screening ,03 medical and health sciences ,Proteorhodopsin ,Bacterial Proteins ,Transferases ,Rhodopsins, Microbial ,medicine ,Escherichia coli ,Translocase ,Cell-free expression ,Lipid bilayer ,MraY translocase ,Nanodisc ,030304 developmental biology ,0303 health sciences ,biology ,Escherichia coli Proteins ,030302 biochemistry & molecular biology ,Nanodiscs ,Membrane Proteins ,Transporter ,Cell Biology ,Membrane integration ,Membrane ,Membrane protein ,biology.protein ,Molecular Chaperones - Abstract
Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.
- Published
- 2012
16. Systems for the cell-free synthesis of proteins
- Author
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Lei, Kai, Christian, Roos, Stefan, Haberstock, Davide, Proverbio, Yi, Ma, Friederike, Junge, Mikhail, Karbyshev, Volker, Dötsch, and Frank, Bernhard
- Subjects
Bacteriophage T7 ,Detergents ,Escherichia coli ,Membrane Proteins ,Magnesium ,DNA ,DNA-Directed RNA Polymerases ,Cell Fractionation ,Protein Engineering ,Recombinant Proteins - Abstract
We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli. Two reaction configurations, batch and continuous exchange, are discussed and analytical scale as well as preparative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide specific protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exemplified as new tools for genomics and proteomics studies.
- Published
- 2011
17. Systems for the Cell-Free Synthesis of Proteins
- Author
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Davide Proverbio, Friederike Junge, Lei Kai, Yi Ma, Frank Bernhard, Stefan Haberstock, Mikhail Karbyshev, Volker Dötsch, and Christian Roos
- Subjects
DNA metabolism ,Membrane protein ,Dna genetics ,Chemistry ,Genomics ,Computational biology ,Cell free ,Proteomics - Abstract
We describe a system for the cell-free expression of proteins based on extracts from Escherichia coli. Two reaction configurations, batch and continuous exchange, are discussed and analytical scale as well as preparative scale setups are documented. Guidelines for the systematic development and optimization of cell-free expression protocols are given in detail. We further provide specific protocols and parameters for the cell-free production of membrane proteins. High-throughput screening applications of CF expression systems are exemplified as new tools for genomics and proteomics studies.
- Published
- 2011
18. Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins
- Author
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Davide Proverbio, Friederike Junge, Frank Bernhard, Susanne Stefer, Stefan Haberstock, Volker Dötsch, and Christian Roos
- Subjects
Models, Molecular ,Functional analysis ,Cell-Free System ,Free protein ,Membrane Proteins ,Bioengineering ,General Medicine ,Computational biology ,Biology ,Cell biology ,Folding (chemistry) ,Membrane protein ,Protein Biosynthesis ,Protein biosynthesis ,Humans ,Molecular Biology ,Function (biology) ,Topology (chemistry) ,Biotechnology - Abstract
Cell-free expression has emerged as a powerful technique to overcome major restrictions of classical in vivo membrane protein production, with sample yields of mgms of protein per ml reaction volume possible in less than a day. The open nature and high versatility of cell-free expression allows a variety of completely new ways to rationally design and optimise expression environments as well as to modulate folding kinetics for membrane proteins independent of their origin, size, topology and function. This article summarises the array of currently available options to modify and develop cell-free expression protocols adapted to the specific requirements of individual membrane proteins. We give further an overview of the recent advances of cell-free production of membrane proteins for structural and functional analysis.
- Published
- 2010
19. Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor
- Author
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Volker Dötsch, Rupert Abele, Davide Proverbio, Birgit Schäfer, Laura M. Luh, Friederike Junge, Frank Bernhard, and Michael Beyermann
- Subjects
Proteomics ,Blotting, Western ,Detergents ,Fluorescence Polarization ,Ligands ,Binding, Competitive ,Chromatography, Affinity ,Cell-free system ,Receptors, G-Protein-Coupled ,chemistry.chemical_compound ,Radioligand Assay ,Structural Biology ,Humans ,Receptor ,G protein-coupled receptor ,Cell-Free System ,Protein Stability ,Circular Dichroism ,Reproducibility of Results ,Receptor, Endothelin A ,Receptor–ligand kinetics ,chemistry ,Biochemistry ,Membrane protein ,Solubility ,TCEP - Abstract
G-protein coupled receptors still represent one of the most challenging targets in membrane protein research. Here we present a strategic approach for the cell-free synthesis of these complex membrane proteins exemplified by the preparative scale production of the human endothelin A receptor. The versatility of the cell-free expression system was used to modulate sample quality by alteration of detergents hence presenting different solubilization environments to the synthesized protein at different stages of the production process. Sample properties after co-translational and post-translational solubilization have been analysed by evaluation of homogeneity, protein stability and receptor ligand binding competence. This is a first quality evaluation of a membrane protein obtained in two different cell-free expression modes and we demonstrate that both can be used for the production of ligand-binding competent endothelin A receptor in quantities sufficient for structural approaches. The presented strategy of cell-free expression protocol development could serve as basic guideline for the production of related receptors in similar systems.
- Published
- 2010
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