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Imre Berger - One of the best experts on this subject based on the ideXlab platform.
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Multiprotein complex production in e coli the secyeg secdfyajc yidc holotranslocon
Methods of Molecular Biology, 2017Co-Authors: Imre Berger, Ian Collinson, Quiyang Jiang, Ryan J Schulze, Christiane SchaffitzelAbstract:A modular approach for balanced overexpression of recombinant Multiprotein Complexes in E. coli is described, with the prokaryotic protein secretase/insertase complex, the SecYEG-SecDFYajC-YidC holotranslocon (HTL), used as an example. This procedure has been implemented here in the ACEMBL system. The protocol details the design principles of the monocistronic or polycistronic DNA constructs, the expression and purification of functional HTL and its association with translating ribosome nascent chain (RNC) Complexes into a RNC-HTL supercomplex.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J FitzgeraldAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J Fitzgerald, Deepak Thimiri Govinda B RajAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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the production of Multiprotein Complexes in insect cells using the baculovirus expression system
Methods of Molecular Biology, 2015Co-Authors: Imre Berger, Kapil Gupta, Frederic Garzoni, Wassim Abdulrahman, Laura Radu, Olga Kolesnikova, Judit Oszpapai, Arnaud PoterszmanAbstract:Abstract The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these Complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of Multiprotein Complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.
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Multiprotein complex production in insect cells by using polyproteins.
Methods in Molecular Biology, 2013Co-Authors: Yan Nie, Itxaso Bellon-echeverria, Simon Trowitzsch, Christoph Bieniossek, Imre BergerAbstract:A powerful approach utilizing polyproteins for balancing stoichiometry of recombinant Multiprotein Complexes overproduced in baculovirus expression vector systems (BEVS) is described. This procedure has been implemented here in the MultiBac system but can also be directly adapted to all commonly used BEVS. The protocol details the design principles of polyprotein-expressing DNA constructs, the generation of composite baculovirus for polyprotein production, and the expression and in vivo processing of polyproteins in baculovirus infected insect cells.
Wolfgang W. A. Schamel - One of the best experts on this subject based on the ideXlab platform.
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Blue native polyacrylamide gel electrophoresis (BN-PAGE) for analysis of Multiprotein Complexes from cellular lysates
Journal of Visualized Experiments, 2010Co-Authors: Gina J. Fiala, Wolfgang W. A. Schamel, Britta BlumenthalAbstract:Multiprotein Complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory Complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Schägger and von Jagow 1991); (Schägger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.
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blue native polyacrylamide gel electrophoresis bn page for the identification and analysis of Multiprotein Complexes
Science Signaling, 2006Co-Authors: Mahima Swamy, Gabrielle M Siegers, Susana Minguet, Bernd Wollscheid, Wolfgang W. A. SchamelAbstract:Multiprotein Complexes (MPCs) play crucial roles in cell signaling. Two kinds of MPCs can be distinguished: (i) Constitutive, abundant MPCs--for example, multisubunit receptors or transcription factors; and (ii) signal-induced, transient, low copy number MPCs--for example, Complexes that form upon binding of Src-homology 2 (SH2) domain-containing proteins to tyrosine-phosphorylated proteins. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is a separation method with a higher resolution than gel filtration or sucrose density ultracentrifugation that can be used to analyze abundant, stable MPCs from 10 kD to 10 MD. In contrast to immunoprecipitation and two-hybrid approaches, it allows the determination of the size, the relative abundance, and the subunit composition of an MPC. In addition, it shows how many different Complexes exist that share a common subunit, whether free monomeric forms of individual subunits exist, and whether these parameters change upon cell stimulation. Here, we give a detailed protocol for the separation of MPCs from total cellular lysates or of prepurified MPCs by one-dimensional BN-PAGE or by two-dimensional BN-PAGE and SDS-PAGE.
Daniel J Fitzgerald - One of the best experts on this subject based on the ideXlab platform.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J FitzgeraldAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J Fitzgerald, Deepak Thimiri Govinda B RajAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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baculovirus expression system for heterologous Multiprotein Complexes
Nature Biotechnology, 2004Co-Authors: Imre Berger, Daniel J Fitzgerald, Timothy J RichmondAbstract:The discovery of large Multiprotein Complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein Complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant Multiprotein production and multigene transfer applications. The identification of novel Multiprotein cellular Complexes has accelerated considerably as a consequence of genome-wide analysis of protein-protein interactions 1,2 , powerful multiple-affinity protein purification methods 3,4 and ultrasensitive mass spectrometry 5,6 . Based on extensive two-hybrid searches, the average number of interaction partners for any given protein is estimated in baker’s yeast to be five to eight 7‐9 . The concept of the cell as a collection of multicomponent protein machines, one for essentially every major process, has thereby emerged 10 . This poses formidable challenges for protein production technologies aimed at molecular level structure and function studies of eukaryotic Multiprotein Complexes with intracellular quantities refractory to large-scale extraction from source. Components of Multiprotein Complexes often display low solubility and activity in isolation, necessitating recombinant technologies that permit simultaneous expression of several genes. Consequently, considerable effort has been invested in generating polycistronic vectors carrying multiple expression cassettes. Recently, a polycistronic vector has been used for expression of a transcription factor complex composed of four subunits in E. coli 11 . Moreover, polycistronic vectors have been recognized as useful for therapeutic gene delivery 12,13 .D NA
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Baculovirus expression system for heterologous Multiprotein Complexes
Nature Biotechnology, 2004Co-Authors: Imre Berger, Daniel J Fitzgerald, Timothy J RichmondAbstract:The discovery of large Multiprotein Complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein Complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant Multiprotein production and multigene transfer applications.
Frederic Garzoni - One of the best experts on this subject based on the ideXlab platform.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J FitzgeraldAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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the multibac baculovirus insect cell expression vector system for producing complex protein biologics
Advances in Experimental Medicine and Biology, 2016Co-Authors: Duygu Sari, Imre Berger, Kapil Gupta, Alice Aubert, Petra Drncova, Frederic Garzoni, Daniel J Fitzgerald, Deepak Thimiri Govinda B RajAbstract:Multiprotein Complexes regulate most if not all cellular functions. Elucidating the structure and function of these complex cellular machines is essential for understanding biology. Moreover, Multiprotein Complexes by themselves constitute powerful reagents as biologics for the prevention and treatment of human diseases. Recombinant production by the baculovirus/insect cell expression system is particularly useful for expressing proteins of eukaryotic origin and their Complexes. MultiBac, an advanced baculovirus/insect cell system, has been widely adopted in the last decade to produce Multiprotein Complexes with many subunits that were hitherto inaccessible, for academic and industrial research and development. The MultiBac system, its development and numerous applications are presented. Future opportunities for utilizing MultiBac to catalyze discovery are outlined.
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the production of Multiprotein Complexes in insect cells using the baculovirus expression system
Methods of Molecular Biology, 2015Co-Authors: Imre Berger, Kapil Gupta, Frederic Garzoni, Wassim Abdulrahman, Laura Radu, Olga Kolesnikova, Judit Oszpapai, Arnaud PoterszmanAbstract:Abstract The production of a homogeneous protein sample in sufficient quantities is an essential prerequisite not only for structural investigations but represents also a rate-limiting step for many functional studies. In the cell, a large fraction of eukaryotic proteins exists as large multicomponent assemblies with many subunits, which act in concert to catalyze specific activities. Many of these Complexes cannot be obtained from endogenous source material, so recombinant expression and reconstitution are then required to overcome this bottleneck. This chapter describes current strategies and protocols for the efficient production of Multiprotein Complexes in large quantities and of high quality, using the baculovirus/insect cell expression system.
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robots pipelines polyproteins enabling Multiprotein expression in prokaryotic and eukaryotic cells
Journal of Structural Biology, 2011Co-Authors: Lakshmi Sumitra Vijayachandran, Simon Trowitzsch, Christoph Bieniossek, Frederic Garzoni, Christiane Schaffitzel, Christophe Romier, Cristina Viola, Maxime Chaillet, Didier Busso, Arnaud PoterszmanAbstract:Multiprotein Complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these Multiprotein Complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their Complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein Complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing Multiprotein Complexes.
Timothy J Richmond - One of the best experts on this subject based on the ideXlab platform.
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automated unrestricted multigene recombineering for Multiprotein complex production
Nature Methods, 2009Co-Authors: Christoph Bieniossek, Philipp Berger, Christiane Schaffitzel, Timothy J Richmond, Daniel Frey, Natacha Olieric, Ian Collinson, Christophe Romier, Michel O Steinmetz, Imre BergerAbstract:Structural and functional studies of many Multiprotein Complexes depend on recombinant-protein overexpression. Rapid revision of expression experiments and diversification of the Complexes are often crucial for success of these projects; therefore, automation is increasingly indispensable. We introduce Acembl, a versatile and automatable system for protein-complex expression in Escherichia coli that uses recombineering to facilitate multigene assembly and diversification. We demonstrated protein-complex expression using Acembl, including production of the complete prokaryotic holotranslocon.
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multibac multigene baculovirus based eukaryotic protein complex production
Current protocols in protein science, 2008Co-Authors: Christoph Bieniossek, Timothy J Richmond, Imre BergerAbstract:Multiprotein Complexes are an emerging focus in current biology, resulting in a demand for advanced heterologous expression systems. This unit provides protocols for the expression of eukaryotic Multiprotein Complexes using multigene expression vectors. Homologous and site-specific recombinases facilitate their assembly. Thus, modification of individual subunits for revised expression studies is achieved with comparative ease. The strategy outlined here employs the MultiBac baculoviral expression system for Multiprotein Complexes as an example. Baculoviral expression does not require particular safety precautions due to the replication incompetence of baculovirus in mammalian hosts. The MultiBac system provides for improved protein production due to deletion of specific viral genes (V-cath, chiA). Most of the steps described in this unit are tailored for high-throughput approaches. The general strategy of rapidly combining encoding DNAs by recombination into multigene expression vectors for protein complex expression can also be applied to other prokaryotic or mammalian expression systems.
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baculovirus expression system for heterologous Multiprotein Complexes
Nature Biotechnology, 2004Co-Authors: Imre Berger, Daniel J Fitzgerald, Timothy J RichmondAbstract:The discovery of large Multiprotein Complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein Complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant Multiprotein production and multigene transfer applications. The identification of novel Multiprotein cellular Complexes has accelerated considerably as a consequence of genome-wide analysis of protein-protein interactions 1,2 , powerful multiple-affinity protein purification methods 3,4 and ultrasensitive mass spectrometry 5,6 . Based on extensive two-hybrid searches, the average number of interaction partners for any given protein is estimated in baker’s yeast to be five to eight 7‐9 . The concept of the cell as a collection of multicomponent protein machines, one for essentially every major process, has thereby emerged 10 . This poses formidable challenges for protein production technologies aimed at molecular level structure and function studies of eukaryotic Multiprotein Complexes with intracellular quantities refractory to large-scale extraction from source. Components of Multiprotein Complexes often display low solubility and activity in isolation, necessitating recombinant technologies that permit simultaneous expression of several genes. Consequently, considerable effort has been invested in generating polycistronic vectors carrying multiple expression cassettes. Recently, a polycistronic vector has been used for expression of a transcription factor complex composed of four subunits in E. coli 11 . Moreover, polycistronic vectors have been recognized as useful for therapeutic gene delivery 12,13 .D NA
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Baculovirus expression system for heterologous Multiprotein Complexes
Nature Biotechnology, 2004Co-Authors: Imre Berger, Daniel J Fitzgerald, Timothy J RichmondAbstract:The discovery of large Multiprotein Complexes in cells has increased the demand for improved heterologous protein production techniques to study their molecular structure and function. Here we describe MultiBac, a simple and versatile system for generating recombinant baculovirus DNA to express protein Complexes comprising many subunits. Our method uses transfer vectors containing a multiplication module that can be nested to facilitate assembly of polycistronic expression cassettes, thereby minimizing requirements for unique restriction sites. The transfer vectors access a modified baculovirus DNA through Cre-loxP site-specific recombination or Tn7 transposition. This baculovirus has improved protein expression characteristics because specific viral genes have been eliminated. Gene insertion reactions are carried out in Escherichia coli either sequentially or concurrently in a rapid, one-step procedure. Our system is useful for both recombinant Multiprotein production and multigene transfer applications.
