Recombinant protein expression in insect cells using the. This manual is supplied with the products listed below. It is based on sitespecific transposition of an expression cassette into a baculovirus shuttle vector bacmid propagated in e. The infected sf9 cells and supernatant for secreted protein will be used to evaluate the target protein expression timely. There are mainly three insect cell lines used in the baculovirus expression system, the s. Bacto bac baculovirus expression system an efficient sitespecific transposition system to generate baculovirus for highlevel expression of recombinant proteins catalog numbers 10359016, 10360014, 10584027, 10712024 document part number 10359 publication number man0000414 revision a. Thus, we created new crisprcas9 vectors that can be used for genome editing in two relevant insect cell lines. Expression of recombinant adenylyl cyclases in insect sf9 cells is performed as described. Monitoring of the effects of transfection with baculovirus on.
Advantages of using the baculovirus expression vector system. A simplified baculovirusaav expression vector system coupled. The gibco expisf system is the first ever baculovirus expression system that includes both a chemically defined medium and protein expression enhancer for reliable results you can count on. Monitoring of the effects of transfection with baculovirus. Guide to baculovirus expression vector systems bevs and. The baculovirus expression system has many desirable features that are undoubtedly responsible for its success.
Recently, baculovirus expression system and sf9 cell line have widely used for recombinant protein production. Expression systems is open and ready to ship expression systems is committed to supporting our customers through this challenging time. Systems based approach to optimize baculovirusbased protein expression. Here, we present data on the performace of a novel sf9 based baculovirus expression system based upon a yeastolatefree, animal originfree, chemicallydefined, highdensity culture medium that allows for sf9 cells to reach densities nearly twice as high as those attained in traditional yeastolatecontaining media. The baculovirus expression vector system bevs is a versatile platform for. The baculovirus expression vector system bev is used to produce high levels of recombinant proteins in insect cells. Sf9 cells, using a baculovirus expression vector asm.
Systems based approach to optimize baculovirus based protein expression. Genetic optimization of the autographa californica nucleopolyhedrovirus acmnpv genome yields recombinant virus in a single step and eliminates the need for separation of recombinant and parental virus by plaque purification. Traditional baculovirus expression systems utilize tedious and time consuming sitespecific transposition in e. Baculovirus titration method based on moi values for. Baculovirus expression systems typically require bacterial transformation and isolation of a large bacmid or cotransfection of a transfer vector and linear baculovirus dna into insect cells. Sf9 insect cells are infected with three different recombinant baculovirus constructs. After the initial publication of the threebaculovirus system10 various other designs have followed figure 1. There are several advantages of using baculovirus expression system over e. More than 600 recombinant genes have been expressed in baculoviruses to date. The baculovirus insect cell expression system is widely used in producing recombinant proteins. The bactobac baculovirus expression system enables the efficient production of recombinant baculovirus for expression testing in insect cells. Because we can only send you a small sample of a plaquepurified, first passage pp1p1 virus stock, you will need to produce and titer a working virus stock before producing your recombinant protein of interest.
Becauseofthe lowabundanceofbcl2in lymphocytes, overexpression ofthis protein in sf9 insect cells will avail large quantities ofproteinforstructural, functional, andbiochemical analysis. Overexpressed fulllength humanbcl2 baculovirusinfected sf9. The establishment of a controllable expression system in. A chemically defined baculovirus based system for enhanced protein and virus production in sf9 cells figure 1. The following are important choices in designing a system for recombinant protein production. Us20140349374a1 baculovirus system for the expression of. Efficient virus titration should be established to achieve optimal multiplicity of infection moi conditions, which are in turn essential for strong expression of the recombinant ga733 fused to the human immunoglobulin igg fc fragment ga733. We use cookies to improve your browsing experience and provide meaningful content.
Pdf baculovirus expression system scaleup by perfusion. Production and purification of baculovirus for gene. Most baculovirus expression systems are also not suited for highlevel secretion of recombinant. Recombinant protein production in baculovirusinsect. Active application number us14234,988 other versions us20140349374a1 en inventor lionel galibert ottowilhelm. Since the development of the baculovirus expression vector system bevs in the 80s, thousands of recombinant proteins. The expisf expression system starter kit provides cells, culture medium, and reagents to infect 1 liter of cell culture. Baculovirus system for the expression of a gene therapy vector download pdf info publication number us10017783b2. Bd baculogold baculovirus expression system innovative. Baculovirus expression vector system linkedin slideshare.
Overview of the baculovirus expression system request pdf. Us10017783b2 baculovirus system for the expression of a. At the end of each infection cycle, most of the cells show cytopathic effect with an increased cellular diameter and dead or lysed cells, which are signs for. Expression systems provides tools to support the baculovirus and host cell expression system including baculovirus vectors and kits to determine baculovirus production. Baculodirect baculovirus expression system for cloning and highlevel expression of recombinant proteins using gatewayadapted baculovirus dna catalog numbers 125620, 12562039, 12562054, 12562062 revision date 4 january 2012 publication part number 250650 man0000377 for research use only. Guide to baculovirus expression vector systems bevs and insect cell culture. Traditional baculovirus expression systems are based on an in vivo cultured insect cells low frequency recombination event between a shuttle vector carrying the gene of interest and the circular autographa californica nuclear. Generation of recombinant baculoviruses and gene expression with the bactobac expression system. A simplified baculovirusaav expression vector system.
Preparing and titering baculovirus working stocks for. Impact of inverted terminal repeat integrity on raav8 production using the baculovirussf9 cells system. The production system most commonly used is based on autographa californica multinuclear polyhedrosis virus acmnpv. The baculovirusinsect cell expression system is widely used in producing recombinant proteins. Because the flashbac system has effectively reduced recombinant baculovirus production to a onestep procedure, it is fully amenable to high throughput and automated production pla tforms. In the present study, the sf9 cell line was transfected with. Recent decades have witnessed the growing use of baculovirus vectors to produce and obtain a range of recombinant proteins 1,2. We have thousands of liters of cell culture media esf 921 and esf af in stock and ready to ship both domestically and internationally.
More recently, it is emerging as a promising vector for gene therapy application 5. This includes the fivebaculovirus system12, the redesigned threebaculovirus system, and the consolidated twobaculovirus system. As with all protein expression systems the baculovirus has its advantages and setbacks, the tradeoff for high levels of expression, safety and posttranslational modifications is often time, with the baculovirus system being more labour intensive than simple bacterial methods. Expression systems insect cell culture media and baculovirus. Baculovirus is primarily used for the production of recombinant proteins and vaccines in lepidopteran spodoptera fugiperda sf9 insect cells by using recombinant autographa californica multicapsid nuclear polyhedrosis virus acmnpv 1, 2, 3, 4. The baculovirus expression vector system bevs is now widely used for. Since 1985, when the first protein il2 was produced in large scale from a recombinant baculovirus, use of bevs has increased. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their.
They are one of the most prominent viruses known to affect the insect population 1. Baculovirus control additional products expression systems offers a variety of media formulations for insect and mammalian cell culture. The bactobac baculovirus expression system the bactobac baculovirus expression system provides a rapid and highly effective method to generate recombinant baculoviruses based on sitespecific transposition of an expression cassette into a baculovirus shuttle vector bacmid propagated in e. Many nonessential genes may be replaced by gene of interest baculovirus expression vector system the resulting recombinant baculovirus lacks one of nonessential gene polh, vcath, chia etc. Preparing and titering baculovirus working stocks for protein production. Crisprcas9 is a powerful sitespecific genomeediting tool that has been used to genetically engineer many different systems. The baculovirus insect cell expression system utilizes recombinant baculoviruses insect viruses and their ability to manufacture high yields of biologically active proteins from infected insect cells. Baculodirect baculovirus expression system thermo fisher. Sf9 or sf21 cells are generally used for cotransfection s, virus. Gateway destination vectors for baculovirus expression system pdf, list.
Recombinant baculovirus are widely used in the expression of heterologous genes in cultured insect cells. This insect cell expression system also utilizes a nonengineered derivative of sf9 insect cells that have been adapted for highdensity suspension growth. The baculovirus expression system offers the following advantages over prokaryotic and other eukaryotic systems. Protein production using the baculovirusinsect cell. For customers who prefer a eukaryotic expression system, are exploring lowercost alternatives to mammalian expression system but do not want to compromise on the overall quality. Specialized media, transfection reagents, and vectors have been developed in response to recent advances in insect cell culture and molecular biology methods. Baculoviruses have emerged as a popular system for overproducing recombinant proteins in eukaryotic cells. Expisf baculovirus expression system thermo fisher.
Nih reference number e0092008 product type research tools keywords immune, inflammation, insect cell line, baculovirus, protein expression sf9et, baculovirus titer, esposito. May 29, 20 recently, baculovirus expression system and sf9 cell line have widely used for recombinant protein production. Oct 12, 2015 in this regard, the baculovirus expression vector system bevs is one of the technologies of choice to generate such highly immunogenic vaccines. The bevs is a potent eukaryotic expression system that can be utilized to express a variety of recombinant proteins at levels as high as 500 mg per liter oreilly et al. Baculovirusinsect cell expression system for beginners introduction baculovirus mediated insect cell expression system is one of the most popular eukaryotic expression systems for protein production. Baculovirus expression system an overview sciencedirect topics. This unit gives an overview of the baculovirus expression system, including discussion of. Recombinant protein production in baculovirusinsect expression.
Recombinant protein expression in insect cells using the baculovirus expression system background. Guide to baculovirus expression vector systems bevs and insect. Examined parameters, moi and infection time, were expanded to determine optimum moi value 0. The baculovirus autographa californica multiple nuclear polyhedrosis virus acmnpv expression system has been extensively used for expression of a wide variety of mammalian genes, including human ones. Bactobac baculovirus expression system thermo fisher scientific. Production and purification of baculovirus for gene therapy. Baculovirusinsect cell expression system for beginners. Improved production efficiency of viruslike particles by the. The system relies on generation of recombinant baculovirus by sitespecific transposition in e. This has been a main workhorse to fulfill high demand of recombinant protein needed for structural, biophysics and biochemical studies. Expression system advantages as with all protein expression systems the baculovirus has its advantages and setbacks, the tradeoff for high levels of expression, safety and posttranslational modifications is often time, with the baculovirus system being more labour intensive than simple bacterial methods. The baculovirus expression vector system the baculovirus expression vector system bevs is one of the most powerful and versatile eukaryotic expression systems available. The baculovirus expression system bevs has become a vital gene.
Jan 27, 2018 efficient virus titration should be established to achieve optimal multiplicity of infection moi conditions, which are in turn essential for strong expression of the recombinant ga733 fused to the human immunoglobulin igg fc fragment ga733. Impact of inverted terminal repeat integrity on raav8. Baculovirus as mammalian cell expression vector for gene. The baculovirus expression system bevs has become a vital gene expression tool in the production and characterization of numerous recombinant proteins. It is known to have a broad host and tissue tropism, infects both. Overview of the baculovirus expression system chambers. Another advantage of bevs is that insect cells possess similar posttranslational modifications processing. No hightiter stocks can be generated from drosophila cells. Improved production efficiency of viruslike particles by. Amplify the resultant recombinant virus in sf9 insect cells. Expression of sindbis virus 26s cdna in spodoptera frugiperda. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed. Baculovirus expression vector system bd biosciences. The gibco expisf expression system is a highyield baculovirusbased insect expression system based on suspensionadapted sf9 spodoptera frugiperda cells.
Insect cellbased recombinant adenoassociated virus. The gibco expisf expression system is a highyield baculovirus based insect expression system based on suspensionadapted sf9 spodoptera frugiperda cells. Baculovirus expression system, bacpak, is a complete system for expressing fullyfunctional recombinant proteins at extremely high levels in insect host cells. A perfusion system based on a 4l stirred tank bioreactor and a customdesigned tangential crossflow filter was assembled to realize a scaleup of the baculovirus expression vector system bevs.
After the initial publication of the three baculovirus system 10 various other designs have followed figure 1. The recombinant virus genome replicates to produce baculovirus that can be harvested directly from the culture medium of transfected insect cells. Here we cover the benefits of working with the baculovirus system in your lab. Pdf baculovirus expression system scaleup by perfusion of. The proteins of interest can be easily purified from infected cells or their supernatants using tag and affinity chromatography. For customers who prefer a eukaryotic expression system, are exploring lowercost alternatives to mammalian expression system but. Nih reference number e0092008 product type research tools keywords immune, inflammation, insect cell line, baculovirus, protein expression sf9 et, baculovirus titer, esposito. The invention concerns a recombinant baculovirus genome useful for the expression of gene therapy vectors by means of a single infection. Baculodirect baculovirus expression system makes baculovirus expression more convenient, requiring less hands on time than traditional systems. New insect sf9et cell line for determining baculovirus titers. A chemically defined baculovirusbased system for enhanced protein and virus production in sf9 cells figure 1. Implementation of the use of complete itrs in the frame of the baculovirusbased raav expression system is one step that will be required to optimize the quality of raavbased gene therapy drugs.
The insectselect system uses the opie2 promoter from the orgyia pseudotsugata multicapsid nucleopolyhedrosis virus for highlevel expression. In addition to the very highlevel expression in insect cells, the bac ulovirus insect expression system provides a eukaryotic. Achieving highyield production of functional aav5 gene. Dec 23, 2015 many nonessential genes may be replaced by gene of interest baculovirus expression vector system the resulting recombinant baculovirus lacks one of nonessential gene polh, vcath, chia etc. However, this tool has not yet been adopted for use in the baculovirusinsect cell system, which is an important recombinant protein production platform. The hdppiv gene was successfully expressed in the baculovirusinsect cell system in a related study dobers et al. Baculovirus supernatant is further amplified in sf9 suspension culture through ongoing infection of freshly added sf9 cells in a shaker flask with serumfree insect cell culture medium. Baculovirus expression systems dna transfection for baculovirus expression vector system 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, bacnblue dna invitrogen, or bacpak6 dna clontech. Based system for enhanced protein and virus production in.