multibac

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...

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...

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...

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...

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...

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...

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...

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...

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...

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...

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...

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...

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...

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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...

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...

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...

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...

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...

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...