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Le T M Le, Jens R Nyengaard, Monika M Golas, Bjoern Sander
Integral membrane proteins play a central role in various cellular functions and are important therapeutic targets. However, technical challenges in the overexpression and purification of membrane proteins often represent a limiting factor for biochemical and structural studies. Here, we constructed a set of vectors, derivatives of MultiBac vectors that can be used to express proteins with a cleavable N-terminal signal peptide in insect cells. We propose these vectors for expression of type I membrane proteins and other secretory pathway proteins that require the signal recognition particle for translocation to the endoplasmic reticulum (ER)...
January 2018: Molecular Biotechnology
Chia-Liang Lin, Yuriy Chaban, David M Rees, Elizabeth A McCormack, Lorraine Ocloo, Dale B Wigley
We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit...
July 7, 2017: Nucleic Acids Research
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
W Zhang, A Aubert, J M Gomez de Segura, M Karuppasamy, S Basu, A S Murthy, A Diamante, T A Drury, J Balmer, J Cramard, A A Watson, D Lando, S F Lee, M Palayret, S L Kloet, A H Smits, M J Deery, M Vermeulen, B Hendrich, D Klenerman, C Schaffitzel, I Berger, E D Laue
The nucleosome remodeling deacetylase (NuRD) complex is a highly conserved regulator of chromatin structure and transcription. Structural studies have shed light on this and other chromatin modifying machines, but much less is known about how they assemble and whether stable and functional sub-modules exist that retain enzymatic activity. Purification of the endogenous Drosophila NuRD complex shows that it consists of a stable core of subunits, while others, in particular the chromatin remodeler CHD4, associate transiently...
July 17, 2016: Journal of 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
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
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
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, 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
Lunguang Yao, Shanshan Wang, Shuo Su, Ning Yao, Jian He, Li Peng, Jingchen Sun
A new baculovirus-silkworm multigene expression system named Bombyx mori MultiBac is developed and described here, by which multiple expression cassettes can be introduced into the Bombyx mori nuclear polyhedrosis virus (BmNPV) genome efficiently. The system consists of three donor vectors (pCTdual, pRADM and pUCDMIG) and an invasive diaminopimelate (DAP) auxotrophic recipient E. coli containing BmNPV-Bacmid (BmBacmid) with a homologous recombination region, an attTn7 site and a loxp site. Two genes carried by pCTdual are firstly inserted into BmBacmid by homologous recombination, while the other eight genes in pRADM and pUCDMIG are introduced into BmBacmid through Tn7 transposition and cre-loxp recombination...
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
Yajing Zhou, Huiqing Chen, Xiao Li, Yujue Wang, Keping Chen, Sufang Zhang, Xiao Meng, Ernest Y C Lee, Marietta Y W T Lee
Eukaryotic DNA polymerase δ (pol δ) plays a crucial role in chromosomal DNA replication and various DNA repair processes. It is thought to consist of p125, p66 (p68), p50 and p12 subunits. However, rigorous isolation of mammalian pol δ from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. While recombinant pol δ isolated from infected insect cells have some problems of consistency in the quality of the preparations, and the yields are much lower. To address these deficiencies, we have constructed recombinant BmNPV baculoviruses using MultiBac system...
2011: PloS One
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
Hu Long, Lun-guang Yao, Shan-shan Wang, Shin-xin Chen, Pei-chan Tan, Jing-chen Sun
OBJECTIVE: To construct recombinant baculoviruses co-expressing three structural genes vp2, vp6 and vp7 of rotavirus, and assemble rotavirus-like particles (VLPs) in BmN cells. METHODS: Human group A rotavirus was cultivated in MA104 cells, and the RNA was extracted and the three genes were obtained by RT-PCR. The PCR products were inserted into the transfer vectors pFBDM and pUCDM, respectively. A enhanced green fluorescent protein gene (egfp) driven by IE1 promoter was introduced into pFBDM to investigate the efficiency of infection...
July 2010: Nan Fang Yi Ke da Xue Xue Bao, Journal of Southern Medical University
Simon Trowitzsch, Christoph Bieniossek, Yan Nie, Frederic Garzoni, Imre Berger
Most eukaryotic proteins exist as large multicomponent assemblies with many subunits, which act in concert to catalyze specific cellular activities. Many of these molecular machines are only present in low amounts in their native hosts, which impede purification from source material. Unraveling their structure and function at high resolution will often depend on heterologous overproduction. Recombinant expression of multiprotein complexes for structural studies can entail considerable, sometimes inhibitory, investment in both labor and materials, in particular if altering and diversifying of the individual subunits are necessary for successful structure determination...
October 2010: Journal of Structural Biology
Tilo Senger, Lysann Schädlich, Lutz Gissmann, Martin Müller
Human papillomavirus (HPV) L1 self-assembles into virus-like particles (VLPs), which are the basis for the two commercially available prophylactic vaccines. For one of them (Cervarix) HPV 16 and 18 VLPs are being produced in insect cells using the baculovirus expression system. However, due to low yield, production of VLPs remains challenging for certain other PV types. Here we report that employment of a modified baculovirus-based (MultiBac) expression system (Berger, I., Fitzgerald, D. J., and Richmond, T...
June 5, 2009: Virology
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