For more information, refer to our Web site at www.invitrogen.com or call Technical Service (see page 28). The Bac -to -Bac Baculovirus Expression system (Invitrogen Life Technologies, Carlsbad, CA, USA) provides a rapid and efficient method for generating recombinant baculoviruses(7). © 2007-2020 Sino Biological Inc. All rights reserved, Common Cytokine Receptor Signaling Pathway, Antibody Development & Antibody Production, • HEK293 / CHO transient expression service, Communicate about protein expression requirements, Carry out pilot study for protein expression, Feedback pilot study result and ship sample (if feasible), Bulk production based on customer requirements, One-stop service from gene sequence to purified protein, High-efficiency expression of intracellular and extracellular proteins. Schematic depiction of the recombinant baculovirus construct and experimental design. Not for use in diagnostic procedures. The Bac-to-Bac™Baculovirus Expression System enables rapid and efficient generation of recombinant baculovirus. Baculovirus Expression of rHiAChE. In contrast, BaculoDirect™ Baculovirus Expression System uses a quick, 1 hour Gateway® recombination reaction to produce the necessary bacmid for transfection, saving days to produce recombinant baculovirus. The baculovirus expression system has been extensively used for the expression of recombinant proteins within insect cells for a number of years. Bac-to-Bac® Baculovirus Expression System An efficient site-specific transposition system to generate baculovirus for high-level expression of recombinant proteins Catalog Numbers 10359-016, 10360-014, 10584-027, 10712-024 Document Part Number … The system offers several advantages,including: Able to perform complex post-translational modifications (PTMs) High success rate of soluble protein recovery; Note: Reagents are available in other sizes. Figure 1. This method is based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (bacmid) propagated in … … Includes: Each Bac-to-Bac Baculovirus Expression System includes 10µg of pFastBac1 vector, 0.5mL MAX Efficiency DH10Bac Chemically Competent Cells, pFastBac 1-Gus control vector, and 1mL of Cellfectin Reagent. Since 1983, when the baculovirus expression vector was introduced (1, 2), the baculovirus expression vector system (BEVS) has become a useful tool for the expression of recombinant proteins in insect cells , and more recently, in vertebrate cells . Given its development speed and versatility for the expression of a wide range of protein families, the BEVS offers multiple advantages for protein production in a variety of applications. Quick rundown of the bac-to-bac system. GenScript's BacuVance baculovirus expression system was developed by our in-house team of scientists for virus production and expression of recombinant proteins from baculovirus-infected insect cells. Seven recombinant baculovirus vectors with different expression cassettes were constructed using the transfer vector pFastBac1 (Invitrogen, Carlsbad, CA; Table 1).Two of them contain the CMV enhancer/promoter to drive expression of a firefly luciferase reporter gene (BV-CMV-Luc) and an enhanced GFP (EGFP) … In addition to attR site for quick Gateway® recombination cloning, the backbone contains strong polyhedrin promoter for high protein expression and a C-terminal or N-terminal 6xHis and V5 tag for detection and purification. Baculovirus expression system for heterologous multiprotein complexes Imre Berger1,2, Daniel J Fitzgerald1,2 & Timothy J Richmond1 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. The system has several unique features which account for its popularity. 2007, 2008). expression system (Bac-to-Bac Baculovirus Expression System, Invitrogen) has been used to express mouse Gαq in SF9 insect cells. Transfection of Sf9 cells with recombinant bacmid DNA Spodoptera frugiperda (Sf9) cells were cultured at 27 °C in Grace’s insect cell culture medium supplemented with 10% heat-inactivated fetal bovine serum, 50 u/ml penicillin, 50 µg/ml streptomycin, with 9×10 5 cells in 2 ml Ten Microliters of the LR reaction was used to transfect 2 x 106 Sf21 cells with Cellfectin® Reagent. Total RNA from heads of adult horn flies either susceptible or resistant to diazinon (containing the HiAChE/G262A mutation) was used as template for oligo(dT 18 V)-primed cDNA synthesis using the SuperScript Choice System (Invitrogen, Carlsbad, CA) as described previously (Temeyer et al. Thermo Fisher Scientific. The baculovirus DNA is from Autogr… Simply mix the Entry clone with the BaculoDirect™ Linear DNA and Gateway® LR Clonase™ enzyme, incubate for 1 hour, and then transfect either Sf9 of Sf21 insect cell to produce recombinant virus. The recombinant baculoviruses (rBacs) were obtained by generating the bacmids by means of the pFastBac1 vector for use with the Bac-To-Bac baculovirus expression system (Invitrogen, Life Technologies). The insect cell-based BEVS has been extensively used to produce many diverse types of recombinant proteins for research, medical, agricultural, and veterinary purposes. Baculovirus is a lytic, large (130 kb), double-stranded DNA virus, and the Autographa californica virus is the most commonly used baculovirus isolate for recombinant expression. The system takes advantage of the site- specific transposition properties of the Tn7 transposon to simplify and enhance the process of generating recombinant bacmid DNA. It is suitable for expressing proteins that are probably harmful to mammalian host cells, such as kinases and toxic proteins. The baculovirus-insect cell expression system is a popular choice for recombinant protein production. Baculovirus-insect cell system belongs to eukaryotic expression system, with capability of protein folding and modification after protein translation. Ten microliters of the resulting supernatant was used to infect 2 x 106 Sf21 cells. The baculovirus expression vector system (BEVS) is one of the most powerful, robust, and versatile eukaryotic expression systems available. Since 1985, when the first protein (IL-2) was produced in large scale from a recombinant baculovirus, use of BEVS has increased dramatically (5). Advantages of the BaculoDirect™ System: BaculoDirect™ Baculovirus Expression System makes baculovirus expression more convenient, requiring less hands on time than traditional systems. I've been working with this system for the past several weeks in my rotation. Invitrogen has developed the Insect Select™ System which uses elements of the baculovirus expression system to express proteins, without needing to produce the baculovirus itself. The anti-gp64 antibody is effective for direct immunofluorescence staining of insect cells for flow cytometric analysis. The results of western blotting experiments showed that SF9 cells infected with Gαq recombinant baculoviruses, express the protein at high levels. After 72 hours, cells were examined for expression by fluorescence. β-gal staining was performed on the cells and no background of non-recombinant virus was performed. A one-hour Gateway® LR reaction was performed using 300 ng of BaculoDirect™ Linear DNA and 100 ng of a Gateway® entry clone containing GFP. For more information about the insect cell lines and Gibco™ cell culture products, see our Web site (www.invitrogen.com) or contact Technical Service (see page 62). Materials and Methods. The insect cell/baculovirus expression vector system (BEVS) is becoming increasingly popular to produce recombinant proteins. Baculovirus-insect cell system belongs to eukaryotic expression system, with capability of protein folding and modification after protein translation. More recently, recombinant baculovirus vectors have been developed to permit transient and stable gene delivery into a number of mammalian cell lines. This plasmid is then introduced into insect cells along with circular wild-type genomic viral DNA. Item Quantity Catalog no. For preparation of total RNA-seq samples, human 293TT and insect Sf9 cells were infected with AcHERV env-CMV-GFP at a MOI of 30 for 72 h (Figure 1B), and total RNA was isolated. Show here at right is western blot analysis of 5 proteins cloned into and expressed using the BaculoDirect™ system. In contrast, BaculoDirect™ Baculovirus Expression System uses a quick, 1 hour Gateway® recombination reaction to produce the necessary bacmid for transfection, saving days to produce recombinant … 12562039,12562013,12562062,12552054,11791023,12562054, 11496015,B82501,12569017,11497013,B82101,12682019,B85502,12552014,10902096,10486025,11605102,25030081, BaculoDirect™ Baculovirus Expression System, Spectroscopy, Elemental & Isotope Analysis, Preclinical to Companion Diagnostic Development, Chromatography Columns, Resins, & Spin Filters, BaculoDirect™ Baculovirus Expression System, Fast method generates recombinant virus in minimal time, Strong polyhedrin promoter produces mgs of protein, C-terminal or N-terminal 6xHis and V5 tag, Flexible Gateway® cloning allows use of any Entry vector. The TB vectors were constructed by cloning the TB cassette into the pFastBac vectors, as described previously . Sino Biological possesses abundant experience of baculovirus-Insect cell protein expression, especially the expression of large proteins, toxic proteins, kinases, intracellular proteins and membrane proteins. More than 600 recombinant genes have been expressed in baculoviruses to date. Recombinant baculovirus vectors. Baculoviridae is a family of viruses. The resultant plasmid (H7N9-TW VLP) was employed to generate the recombination baculovirus for influenza VLP expression using the Bac-to-Bac® system (Invitrogen) [ 11 ]. The recombination baculovirus was successfully established in a BEVS. The BaculoDirect™ system accelerates research by eliminating these time-consuming steps, saving you precious hands-on time. Post-translational modifications, production of protein complexes, and reported high protein yields are some of the favorable features of this eukaryotic expression system. Cells were again grown in Grace's Medium supplemented with 10% FBS and 100 µM ganciclovir. vii Accessory Products Introduction The products listed in this section are intended for use with the Baculovirus Expression System with Gateway® Technology. A number of pFastBac™ vectors, including pFastBac 1, HT and Dual, may be used in this system… baculovirus expression system and was further propag ated in Sf9 cells. Bac-to-Bac® Baculovirus Expression System From Invitrogen. Invitrogen to facilitate baculovirus-mediated expression of your recombinant protein in insect cells. Introduction The Bac-to-Bac®Baculovirus Expression System is a rapid and efficient method to generate recombinant baculoviruses. Traditional baculovirus expression systems utilize tedious and time consuming site-specific transposition in E. coli or lengthy homologous recombination in insect cells to generate the recombinant baculovirus. Search Alternatively, insect cells are transfected with a recombinant bacmid DNA constructed by transposition of the donor plasmid DNA in E. coli cells, the so-called Bac-to-Bac™ (Invitrogen-Gibco/Life Technologies) method. ( A) For Research Use Only. It is suitable for expressing proteins that are probably harmful to mammalian host cells, such as kinases and toxic proteins. Arthropods, lepidoptera, hymenoptera, diptera, and decapoda serve as natural hosts. baculovirus expression system (Invitrogen Inc.). The BaculoDirect™ linear DNA is designed for simple generation of recombinant baculovirus and expression insect cells. This expression system is ideal for the study of toxic proteins and glycoproteins (which require a secretion signal to be glycosylated). Spodoptera frugiperda (Sf9) insect cells are cotransfected with the transfer vector (donor or shuttle) plasmid DNA containing the foreign gene to be expressed and BaculoGold™ DNA (PharMingen), Bac-N-Blue™ DNA (Invitrogen), or BacPAK6™ DNA (Clontech). The gene to be expressed is first cloned into a plasmid transfer vector downstream from a baculovirus promoter that is flanked by baculovirus DNA derived from a nonessential locus, usually the polyhedrin gene. Also, glycoproteins secreted from baculoviruses can be easily de-glycosylated in vitro —an important feature for protein crystallization. Cells were grown 72 hours in Grace's Medium supplemented with 10% FBS and 100 µM ganciclovir. The baculovirus gp64 envelope glycoprotein is a major component of the envelope of the budded baculovirus and is essential for entry into cultured insect host cells. 241000701447 unidentified baculovirus Species 0.000 title claims description 138 230000014509 gene expression Effects 0.000 title description 30 210000004027 cells Anatomy 0.000 claims description 54 Our engineered BaculoDirect™ linear DNA contains attR sites for recombination of your gene of interest cloned into a Gateway® Entry clone. Traditional baculovirus expression systems utilize tedious and time consuming site-specific transposition in E. coli or lengthy homologous recombination in insect cells to generate the recombinant baculovirus. baculovirus system has become one of the most versatile and powerful eukaryotic vector systems for recombinant protein expression (4). Baculovirus expression systems typically require bacterial transformation and isolation of a large bacmid or co-transfection of a transfer vector and linear baculovirus DNA into insect cells. Invitrogen has developed the Bac-to-Bac® Baculovirus Expression System for scientists who have chosen insect cells as their protein expression system.