A novel baculovirus vector for the production of nonfucosylated recombinant glycoproteins in insect cells (original) (raw)
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Production of therapeutic proteins with baculovirus expression system in insect cell
Entomological Research, 2008
Recombinant DNA technology has a major advantage in that it is capable of producing specific therapeutic proteins on demand in a heterologous expression system. The extent of this notion can be understood when one considers how crucial such proteins are, and how problematic the economical and safe production of such proteins are. Therapeutic recombinant protein production is a fundamental aspect of 21st century biotechnology industries. The improved therapeutic recombinant protein expression systems that use prokaryotic and eukaryotic cells have enabled the development of a multi-billion dollar industry. Among the variety of available heterologous expression systems, the baculovirus-based insect cell expression system has been utilized frequently for the high-level production of therapeutic recombinant proteins. Thus, the baculovirus expression system has been recognized as one of the most powerful expression technologies for production, by virtue of the achievable amount and purity, and the ease of the eukaryotic production process. The majority of therapeutic proteins are glycoproteins originating from humans. The insect-based expression system harbors glycosylation processing pathways, which constitute an advantage over other prokaryotic systems that lack glycosylation. However, there are several drawbacks which must be circumvented in order to establish an efficient system for the production of recombinant proteins. This review presents a brief overview of the perspective, particularly the glycosylation aspect, of the production of therapeutic recombinant proteins via a baculovirus-based insect cell expression system.
Glycobiology, 1998
The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8-aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging.
Glycoconjugate journal, 1999
Glycosylation, the most extensive co- and post-translational modification of eukaryotic cells, can significantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine beta-1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the cu...
Baculovirus-free insect cell expression system for high yield antibody and antigen production
Scientific Reports, 2020
Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Ex...
Insect cells as hosts for the expression of recombinant glycoproteins
Glycoconjugate journal, 1999
Baculovirus-mediated expression in insect cells has become well-established for the production of recombinant glycoproteins. Its frequent use arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. In addition to Spodoptera frugiperda Sf9 cells, which are probably the most widely used insect cell line, other mainly lepidopteran cell lines are exploited for protein expression. Recombinant baculovirus is the usual vector for the expression of foreign genes but stable transfection of - especially dipteran - insect cells presents an interesting alternative. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells. With regard to protein folding and post-translational processing, insect cells are second only...
Novel Insect Cell Line Capable of Complex N‐Glycosylation and Sialylation of Recombinant Proteins
Biotechnology Progress, 2003
Paucimannose or oligomannose structures are usually attached to glycoproteins produced by insect cells, while mammalian glycoproteins usually have complex glycans. The lack of complex glycosylation has limited the use of the insect cell baculovirus expression vector system (BEVS), despite its high productivity and versatility. The availability of cell lines capable of complex glycosylation can overcome such a problem and potentially increase the utility of BEVS. In this work the capability of two novel cell lines, one from Pseudaletia unipuncta (A7S) and one from Danaus plexippus (DpN1), to produce and glycosylate a recombinant protein (secreted human placental alkaline phosphatase, SeAP) was assessed. SeAP produced by Tn5B1–4 cells at a low passage number (<200) was utilized for comparison. The optimal conditions for the production of SeAP by DpN1 cells were defined, and the glycosylation profiles of SeAP produced by the cell lines were quantitatively determined. Both the A7S an...
Glycoconjugate J, 1999
Glycosylation, the most extensive co-and post-translational modi®cation of eukaryotic cells, can signi®cantly affect biological activity and is particularly important for recombinant glycoproteins in human therapeutic applications. The baculovirus-insect cell expression system is a popular tool for the expression of heterologous proteins and has an excellent record of producing high levels of biologically active eukaryotic proteins. Insect cells are capable of glycosylation, but their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. A previous study demonstrated that an immediate early recombinant baculovirus could be used to extend the insect cell N-glycosylation pathway by contributing bovine b-1,4 galactosyltransferase (GalT) immediately after infection. Lectin blotting assays indicated that this ectopically expressed enzyme could transfer galactose to an N-linked glycan on a foreign glycoprotein expressed later in infection. In the current study, glycans were isolated from total Sf-9 cell glycoproteins after infection with the immediate early recombinant baculovirus encoding GalT,¯uorescently conjugated and analyzed by electrophoresis in combination with exoglycosidase digestion. These direct analyses clearly demonstrated that Sf-9 cells infected with this recombinant baculovirus can synthesize galactosylated N-linked glycans.
Baculovirus expression systems for recombinant protein production in insect cells
Recent Patents on …, 2009
Baculoviruses are lethal pathogens of insects, predominantly of the order Lepidoptera. These viruses have a biphasic life cycle, which greatly facilitates their use for biotechnological applications. They were exploited initially as biocontrol agents, and then engineered as protein expression vectors. The baculovirus expression vector system (BEVS) is now widely used for recombinant protein production. More recently they have become a popular choice for development as gene delivery and expression vectors in mammalian cells. This article reviews some of the major developments and patents relating to baculoviruses since their initial use as an expression tool and investigates current technologies alleviating bottlenecks in recombinant gene expression in insect cells.
Thirty years of baculovirus-insect cell protein expression: from dark horse to mainstream technology
Journal of General Virology, 2014
In December 1983, a seminal paper appeared on the overexpression of human IFN-b in insect cells with a genetically engineered baculovirus. The finding that baculoviruses produced massive amounts of two proteins (polyhedrin and p10) by means of two very strong promoters and that the corresponding genes were dispensable for virus propagation in insect cells was crucial in the development of this expression system. During the next 30 years, major improvements were achieved over the original baculovirus expression vector (BEV) system, facilitating the engineering of the baculovirus vectors, the modification of the sugar moieties of glycoproteins expressed in insect cells and the scale-up of the cell culture process. To date, thousands of recombinant proteins have been produced in this successful expression system, including several proteinbased human and veterinary vaccines that are currently on the market. Viral vectors based on adeno-associated virus are being produced using recombinant baculovirus technology and the first gene therapy treatment based on this method has been registered. Specially adapted BEVs are used to deliver and express heterologous genes in mammalian cells, and they may be used for gene therapy and cancer treatment in the future. The purpose of this review is to highlight the thirtieth 'anniversary' of this expression system by summarizing the fundamental research and major technological advances that allowed its development, whilst noting challenges for further improvements.
Recombinant Protein Expression in the Baculovirus-Infected Insect Cell System
Methods in Molecular Biology, 2011
Over the last two decades, the use of eukaryotic cells for expression of recombinant proteins has become the preferred choice for many applications. This is primarily the case when posttranslational modifications and correct disulfide-bond formation are necessary for protein folding and activity. Among the eukaryotic expression systems, the baculovirus-infected insect cell platform has gained particular attention, resulting in the development and implementation of multiple strategies for protein expression. Here, we present baculovirus-infected insect cells as an efficient expression system for eukaryotic proteins. We demonstrate a simplified and a shortened procedure for recombinant virus production that is sufficient for large-scale production of proteins in insect cells.