Read by QxMD icon Read


shared collection
18 papers 0 to 25 followers
May Khanna, Tsuyoshi Imasaki, Vimbai M Chikwana, Samantha Perez-Miller, Gerald O Hunter, Amber Mosley, Yuichiro Takagi, Thomas D Hurley
We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression...
August 2013: Protein Expression and Purification
Dieter Palmberger, Dubravko Rendic
Observed different glycosylation patterns of glycoconjugates (recombinantly) produced in various eukaryotic organisms are a direct consequence of differences in numerous proteins involved in biosynthesis of the relevant glycan chains in these species. The need for efficient, robust and flexible methods for recombinant expression of proteins is met by the recently described MultiBac technology, an advanced and optimized baculovirus-based system for simultaneous recombinant protein expression in insect cells...
2015: Methods in Molecular Biology
Ziguo Zhang, Jing Yang, David Barford
The capacity to reconstitute complex biological processes in vitro is a crucial step in providing a quantitative understanding of these systems. It provides material for structural, biochemical and biophysical analyses and allows the testing of biological hypotheses and the introduction of chemical probes and tags for single molecule analysis. Reconstitution of these systems requires access to homogenous components, usually through their over-production in heterologous over-expression systems. Here we describe the application of the USER (Uracil-Specific Excision Reagent) ligation-free cloning method to assemble recombinant MultiBac transfer vectors for the generation of recombinant baculovirus suitable for the expression of multi-protein complexes in insect cells...
February 15, 2016: Methods: a Companion to Methods in Enzymology
Duygu Sari, Kapil Gupta, Deepak Balaji Thimiri Govinda Raj, Alice Aubert, Petra Drncová, Frederic Garzoni, Daniel Fitzgerald, Imre Berger
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...
2016: Advances in Experimental Medicine and Biology
Daniel J Fitzgerald, Philipp Berger, Christiane Schaffitzel, Kazuhiro Yamada, Timothy J Richmond, Imre Berger
Elucidation of the molecular basis of protein-interaction networks, in particular in higher eukaryotes, is hampered by insufficient quantities of endogenous multiprotein complexes. Present recombinant expression methods often require considerable investment in both labor and materials before multiprotein expression, and after expression and biochemical analysis these methods do not provide flexibility for expressing an altered multiprotein complex. To meet these demands, we have recently introduced MultiBac, a modular baculovirus-based system specifically designed for eukaryotic multiprotein expression...
December 2006: Nature Methods
David Barford, Yuichiro Takagi, Patrick Schultz, Imre Berger
Most essential functions in eukaryotic cells are catalyzed by complex molecular machines built of many subunits. To fully understand their biological function in health and disease, it is imperative to study these machines in their entirety. The provision of many essential multiprotein complexes of higher eukaryotes including humans, can be a considerable challenge, as low abundance and heterogeneity often rule out their extraction from native source material. The baculovirus expression vector system (BEVS), specifically tailored for multiprotein complex production, has proven itself to be uniquely suited for overcoming this impeding bottleneck...
June 2013: Current Opinion in Structural Biology
Anne Riss, Elisabeth Scheer, Mathilde Joint, Simon Trowitzsch, Imre Berger, László Tora
Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus specific transcription. GCN5 (KAT2A) is a member of the GNAT (Gcn5-related N-acetyltransferase) family of HATs. In metazoans this enzyme is found in two functionally distinct coactivator complexes, SAGA (Spt Ada Gcn5 acetyltransferase) and ATAC (Ada Two A-containing). These two multiprotein complexes comprise complex-specific and shared subunits, which are organized in functional modules. The HAT module of ATAC is composed of GCN5, ADA2a, ADA3, and SGF29, whereas in the SAGA HAT module ADA2b is present instead of ADA2a...
November 27, 2015: Journal of Biological Chemistry
Francisca Monteiro, Vicente Bernal, Maxime Chaillet, Imre Berger, Paula M Alves
The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression...
September 10, 2016: Journal of Biotechnology
Imre Berger, Roslyn M Bill
No abstract text is available yet for this article.
June 2015: Current Opinion in Structural Biology
Maysam Mansouri, Itxaso Bellon-Echeverria, Aurélien Rizk, Zahra Ehsaei, Chiara Cianciolo Cosentino, Catarina S Silva, Ye Xie, Frederick M Boyce, M Wayne Davis, Stephan C F Neuhauss, Verdon Taylor, Kurt Ballmer-Hofer, Imre Berger, Philipp Berger
Multigene delivery and subsequent cellular expression is emerging as a key technology required in diverse research fields including, synthetic and structural biology, cellular reprogramming and functional pharmaceutical screening. Current viral delivery systems such as retro- and adenoviruses suffer from limited DNA cargo capacity, thus impeding unrestricted multigene expression. We developed MultiPrime, a modular, non-cytotoxic, non-integrating, baculovirus-based vector system expediting highly efficient transient multigene expression from a variety of promoters...
2016: Nature Communications
Yan Nie, Itxaso Bellon-Echeverria, Simon Trowitzsch, Christoph Bieniossek, Imre Berger
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...
2014: Methods in Molecular Biology
Imre Berger, Frederic Garzoni, Maxime Chaillet, Matthias Haffke, Kapil Gupta, Alice Aubert
Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.(1,2) Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis...
2013: Journal of Visualized Experiments: JoVE
Liu Liu, Huifang Song, Lei Zhang, Xiaoting Fan, Qian Zhang, Keping Chen, Huiqing Chen, Yajing Zhou
Bombyx mori nucleopolyhedrovirus (BmNPV) is a major viral agent that causes deadly grasserie disease in silkworms. BmNPV DNA polymerase (Bm-DNAPOL), encoded by the ORF53 gene, plays a central role in viral DNA replication. In this work, a His-tagged Bm-DNAPOL fusion protein, constructed using a novel MultiBac expression system, was overexpressed in Sf-9 insect cells, purified to near homogeneity on Ni-NTA agarose beads and further purified by ion-exchange chromatography. About 0.4 mg of enzyme was obtained from about 1 × 10(9) infected Sf-9 cells in suspension culture...
December 2013: Archives of Virology
Dieter Palmberger, Miriam Klausberger, Imre Berger, Reingard Grabherr
The baculovirus/insect cell system has proven to be a powerful tool for the expression of eukaryotic proteins. Therapeutics, especially in the field of vaccinology, are often composed of several different protein subunits. Conventional baculoviral expression schemes largely lack efficient strategies for simultaneous multi-gene expression. The MultiBac technology which is based on an engineered genome of Autographa californica nuclear polyhedrosis virus in combination with specially designed transfer vectors is an elegant way for flexible generation of multi-subunit proteins in insect cells...
March 2013: Bioengineered
Simon Trowitzsch, Dieter Palmberger, Daniel Fitzgerald, Yuichiro Takagi, Imre Berger
Recombinant production of multiprotein complexes is an emerging focus in academic and pharmaceutical research and is expected to play a key role in addressing complex biological questions in health and disease. Here we describe MultiBac, a state-of-the-art eukaryotic expression technology utilizing an engineered baculovirus to infect insect cells. The robust and flexible concept of MultiBac allows for simultaneous expression of multiple proteins in a single cell, which can be used to produce protein complexes and to recapitulate metabolic pathways...
August 2012: Expert Review of Proteomics
Yajing Zhou, Xiao Meng, Sufang Zhang, Ernest Y C Lee, Marietta Y W T Lee
Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol...
2012: PloS One
Christoph Bieniossek, Tsuyoshi Imasaki, Yuichiro Takagi, Imre Berger
Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes...
February 2012: Trends in Biochemical Sciences
Lakshmi Sumitra Vijayachandran, Cristina Viola, Frederic Garzoni, Simon Trowitzsch, Christoph Bieniossek, Maxime Chaillet, Christiane Schaffitzel, Didier Busso, Christophe Romier, Arnaud Poterszman, Timothy J Richmond, Imre Berger
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...
August 2011: Journal of Structural Biology
Fetch more papers »
Fetching more papers... Fetching...
Read by QxMD. Sign in or create an account to discover new knowledge that matter to you.
Remove bar
Read by QxMD icon Read

Search Tips

Use Boolean operators: AND/OR

diabetic AND foot
diabetes OR diabetic

Exclude a word using the 'minus' sign

Virchow -triad

Use Parentheses

water AND (cup OR glass)

Add an asterisk (*) at end of a word to include word stems

Neuro* will search for Neurology, Neuroscientist, Neurological, and so on

Use quotes to search for an exact phrase

"primary prevention of cancer"
(heart or cardiac or cardio*) AND arrest -"American Heart Association"