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Recombinant baculovirus

Tatsuro Nakamura, Naho Yamaguchi, Yuu Miyauchi, Tomoki Takeda, Yasushi Yamazoe, Kiyoshi Nagata, Peter I Mackenzie, Hideyuki Yamada, Yuji Ishii
Our previous studies have demonstrated functional protein-protein interactions between cytochrome P450 (CYP) 3A and UDP-glucuronosyltransferase (UGT). However, the role of carbohydrate chains of UGTs in the interaction with CYP is not well understood. To address this issue, we examined whether CYP3A1 modulates the function of UGT2B3 which lacks potential glycosylation sites. We also examined whether the introduction of N-glycosylation to UGT2B3 affects CYP3A-dependent modulation of UGT function. To introduce a potential glycosylation site into UGT2B3, Ser 316 of UGT2B3 was substituted with Asn by site-directed mutagenesis...
2016: Frontiers in Pharmacology
Jianxiang Qiu, Zhimin Tang, Meijin Yuan, Wenbi Wu, Kai Yang
During baculovirus infection, most viral proteins must be imported to the nucleus to support virus multiplication. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) orf34 (ac34) is an alphabaculovirus unique gene that is required for optimal virus production. Ac34 distributes in both the cytoplasm and the nuclei of virus-infected Sf9 cells, but contains no conventional nuclear localization signal (NLS). In this study, we investigated the nuclear targeting domains in Ac34. Transient expression assays showed that Ac34 localized in both the cytoplasm and the nuclei of Sf9 cells, indicating that no viral protein is required for Ac34 nuclear localization...
November 25, 2016: Virus Research
María Emilia Smith, Alexandra Marisa Targovnik, Julieta Cerezo, María Alejandra Morales, María Victoria Miranda, Julián Rodríguez Talou
Dengue incidence has grown dramatically in the last years, with about 40% of the world population at risk of infection. Recently, a vaccine developed by Sanofi Pasteur has been registered, but only in a few countries. Moreover, specific antiviral drugs are not available. Thus, an efficient and accurate diagnosis is important for disease management. To develop a low-cost immunoassay for dengue diagnosis, in the present study we expressed the envelope protein domain III of dengue virus type 2 in Rachiplusia nu larvae by infection with a recombinant baculovirus...
November 22, 2016: Protein Expression and Purification
Anping Wang, Yongjuan Wang, Shuang Wu, Weiyong Zuo, Changming Guo, Weiming Hong, Shanyuan Zhu
Recombinant avian adeno-associated virus (rAAAV) is a promising gene transfer vector for avian cells. Although rAAAV can be produced by co-transfection of HEK293 cells with three plasmids, both scalability and productivity of the transient transfection method can not meet the demand for large-scale in vivo experiments. In this study, a scalable rAAAV production method was established by using insect cell/baculovirus expression system. Three recombinant baculoviruses, namely BacARep, BacAVP and BacAGFP, were generated by transfection of Sf9 cells with the three plasmids expressing AAAV Rep genes, modified VP gene or the inverted terminal repeats-flanked green fluorescent protein (GFP) gene...
November 17, 2016: Journal of Virological Methods
Chare Li Kueh, Chean Yeah Yong, Seyedehsara Masoomi Dezfooli, Subha Bhassu, Soon Guan Tan, Wen Siang Tan
Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self-assembled into virus-like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In the current study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self-assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter...
November 14, 2016: Biotechnology Progress
Morikazu Imamura, Naoka Tabeta, Nobuko Kato, Yuichi Matsuura, Yoshifumi Iwamaru, Takashi Yokoyama, Yuichi Murayama
The precise mechanism underlying the conversion of normal prion protein (PrP(C)) into abnormal prion protein (PrP(Sc)) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrP(Sc), results in PrP(Sc) replication that preserves the strain-specific characteristics of the input PrP(Sc); thus, PMCA mimics the process of in vivo PrP(Sc) replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrP(Sc) amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrP(Sc) replication in vitro...
November 7, 2016: Journal of Biological Chemistry
Henna Kekki, Mari Peltola, Sandra van Vliet, Chris Bangma, Yvette van Kooyk, Kim Pettersson
OBJECTIVES: The objective was to study the differences in PSA fucosylation obtained from LNCaP and PC-3 prostate cancer cell lines, seminal plasma PSA and recombinant precursor form of PSA expressed in baculovirus, using Aleuria aurantia lectin (AAL). The aim was to assess whether differences in fucosylation (Fucα1-6/3GlcNAc carbohydrates) of PSA either in urine, blood or tissue enable the discrimination of patients with prostate cancer (PCa) from benign prostatic hyperplasia (BPH) and young males...
November 3, 2016: Clinical Biochemistry
Xiaojun Li, Ran Zhao, Wei Lin, Chenxi Li, Tingting Zhang, Fanyi Meng, Ming Liu, Yun Zhang
The VP1 protein of the foot-and-mouth disease virus (FMDV) is a major target of neutralizing antibodies and is responsible for viral attachment to permissive cells via an RGD motif. VP1 of duck hepatitis A type 1 virus (DHAV-1) does not contain any RGD motif. To investigate the antibody and receptor-binding properties of DHAV-1, VP1 has been expressed as a His fusion protein (His-VP1) in baculovirus system. Sera against His-VP1 raised in rabbits effectively neutralized DHAV-1 infection in vitro and in vivo...
January 2, 2017: Virus Research
V Cunha, K Burkhardt-Medicke, P Wellner, M M Santos, P Moradas-Ferreira, T Luckenbach, M Ferreira
Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish (Danio rerio)...
October 28, 2016: Ecotoxicology and Environmental Safety
Marcia Oliveira Cavalcanti, Eric Vaughn, Ilaria Capua, Giovanni Cattoli, Calogero Terregino, Timm Harder, Christian Grund, Carlos Vega, Francisco Robles, Julio Franco, Ayub Darji, Abdel-Satar Arafa, Egbert Mundt
The evolution of highly pathogenic H5N1 avian influenza viruses (HPAI-H5N1) has resulted in the appearance of a number of diverse groups of HPAI-H5N1 based on the presence of genetically similar clusters of their haemagglutinin sequences (clades). An H5 antigen encoded by a recombinant baculovirus and expressed in insect cells, was used for oil-emulsion-based vaccine prototypes. In several experiments, vaccination was performed at 10 days of age, followed by challenge infection on day 21 post vaccination (PV) with HPAI-H5N1 clades 2...
November 3, 2016: Avian Pathology: Journal of the W.V.P.A
Benjamin Morin, Bo Liang, Erica Gardner, Robin A Ross, Sean P J Whelan
: We report an in vitro RNA synthesis assay for the RNA dependent RNA polymerase (RdRP) of rabies virus (RABV). We expressed RABV large polymerase protein (L) in insect cells from a recombinant baculovirus vector and the phosphoprotein cofactor (P) in Escherichia coli and purified the resulting proteins by affinity and size exclusion chromatography. Using chemically synthesized short RNA corresponding to the first 19-nt of the rabies virus genome we demonstrate that L alone initiates synthesis on naked RNA, and that P serves to enhance initiation and processivity of the RdRP...
October 19, 2016: Journal of Virology
Xiaoyan Chen, Po-Han Chen, Xiaolin He
Semaphorins and their receptor plexins are large glycoproteins that are difficult to express using regular recombinant methods, and the widely used E. coli and baculovirus-insect cell systems have been inadequate for semaphorins and plexins which contain a large number of domains and are heavily modified by glycosylation. Here, we describe the expression of class 7 semaphorin (Sema7A) and the extracellular domain of its receptors PlexinC1, using the baculovirus-mediated mammalian cell gene transduction (BacMam) method...
2017: Methods in Molecular Biology
Christine Koehler, Paul F Sauter, Mirella Wawryszyn, Gemma Estrada Girona, Kapil Gupta, Jonathan J M Landry, Markus Hsi-Yang Fritz, Ksenija Radic, Jan-Erik Hoffmann, Zhuo A Chen, Juan Zou, Piau Siong Tan, Bence Galik, Sini Junttila, Peggy Stolt-Bergner, Giancarlo Pruneri, Attila Gyenesei, Carsten Schultz, Moritz Bosse Biskup, Hueseyin Besir, Vladimir Benes, Juri Rappsilber, Martin Jechlinger, Jan O Korbel, Imre Berger, Stefan Braese, Edward A Lemke
We present a baculovirus-based protein engineering method that enables site-specific introduction of unique functionalities in a eukaryotic protein complex recombinantly produced in insect cells. We demonstrate the versatility of this efficient and robust protein production platform, 'MultiBacTAG', (i) for the fluorescent labeling of target proteins and biologics using click chemistries, (ii) for glycoengineering of antibodies, and (iii) for structure-function studies of novel eukaryotic complexes using single-molecule Förster resonance energy transfer as well as site-specific crosslinking strategies...
October 17, 2016: Nature Methods
Margit Fuchs, Marie-Chloé Boulanger, Herman Lambert, Jacques Landry, Josée N Lavoie
Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes...
2016: Journal of Visualized Experiments: JoVE
Cheuk Wun Li, Roman Osman, Francesca Menconi, Erlinda S Concepcion, Yaron Tomer
Autoimmune polyglandular syndrome 3 variant (APS3v) refers to the co-occurrence of autoimmune thyroiditis (AITD) and type 1 diabetes (T1D) within the same individual. HLA class II confers the strongest susceptibility to APS3v. We previously identified a unique amino acid signature of the HLA-DR pocket (designated APS3v HLA-DR pocket) that predisposes to APS3v. We hypothesized that both thyroid and islet peptides can be presented by the unique APS3v HLA-DR pocket, triggering AITD + T1D together. To test this hypothesis we screened islet and thyroid peptides for their ability to bind to the APS3v HLA-DR pocket...
September 23, 2016: Journal of Autoimmunity
Qi Qi, Lunguang Yao, Zhisheng Liang, Donghua Yan, Zhuo Li, Yadong Huang, Jingchen Sun
Human type II collagen is a macromolecular protein found throughout the human body. The baculovirus expression vector system is one of the most ideal systems for the routine production and display of recombinant eukaryotic proteins in insect, larvae, and mammalian cells. We use this system to express a full-length gene, human type II collagen cDNA (4257 bp), in cultured Spodoptera frugiperda 9 cells (Sf9), Bombyx mori cells, and silkworm larvae. In this study, the expression of human type II collagen gene in both insect cells and silkworm larvae was purified by nickel column chromatography, leading to 300-kDa bands in SDS-PAGE and western blotting indicative of collagen α-chains organized in a triple-helical structure...
September 26, 2016: Molecular Genetics and Genomics: MGG
Y Zhang, Y Shi, H Yu, J Li, Y Quan, T Shu, Z Nie, Y Zhang, W Yu
Baculoviridae is a family of invertebrate viruses with large double-stranded DNA genomes. Proteins encoded by some late expression factor (lef ) genes are involved in the regulation of viral gene expression. Lef-9 is one of four transcription-specific Lefs, which are components of the virus-encoded RNA polymerase, and can initiate and transcribe late and very late genes. As a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus (BmNPV), Lef-9 may be involved in the regulation of viral propagation...
2016: Acta Virologica
Yanyan Miao, Aihua Liang, Yuejun Fu
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) possesses p35 gene, which encodes antiapoptosis protein P35 (or Ac-P35). A previous study showed that deleting p35 promoted premature cytolysis and caused the reduction of virus yield. In this report, we examined the role of P35 in regulating the expression of Ac-IAPs (inhibitor of apoptosis proteins in AcMNPV) and SfP53 (an apoptosis protein in Sf9 cells), and its effect on the production of progeny virus. Results showed that the overexpression of P35 caused a delay in the increase process of SfP53 before 36-h post infection and improved the transcription levels of iaps gene dramatically; it was more favorable to improve the transcription level of iap1 at 24-72 h post infection...
September 15, 2016: Molecular and Cellular Biochemistry
Jennifer L Mehalko, Dominic Esposito
One of the most common methods for producing recombinant baculovirus for insect cell protein production involves a transposition mediated system invented over 2 decades ago. This Tn7-mediated system, commercially sold as Bac-to-Bac, has proven highly useful for construction of high quality baculovirus, but suffers from a number of drawbacks which reduce the efficiency of the process and limit its utility for high throughput protein production processes. We describe here the creation of Bac-2-the-Future, a 2nd generation Tn7-based system for recombinant baculovirus production which uses optimized expression vectors, new E...
November 20, 2016: Journal of Biotechnology
Quan Nguyen, Trinh Tb Tran, Leslie Cl Chan, Lars K Nielsen, Steven Reid
Baculoviruses are recognized as viral workhorses of biotechnology, being used for production of vaccines, complex recombinant proteins, gene delivery vectors' and safe biological pesticides. Improving production yields and understanding the interactions of the virus and its host cell are important aspects of ensuring baculovirus-based processes are commercially competitive. This study aims at potential optimization of host cells used in in vitro virus production by systemically investigating changes in host gene expression in response to virus replication and transcription inside host cells...
November 2016: Applied Microbiology and Biotechnology
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