RNA interference technology to improve the baculovirus-insect cell expression system (original) (raw)
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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.
Generation of baculovirus vectors for the high-throughput production of proteins in insect cells
Biotechnology and Bioengineering, 2008
The baculovirus expression system is one of the most popular methods used for the production of recombinant proteins but has several complex steps which have proved inherently difficult to adapt to a multi-parallel process. We have developed a bacmid vector that does not require any form of selection pressure to separate recombinant virus from non-recombinant parental virus. The method relies on homologous recombination in insect cells between a transfer vector containing a gene to be expressed and a replication-deficient bacmid. The target gene replaces a bacterial replicon at the polyhedrin loci, simultaneously restoring a virus gene essential for replication. Therefore, only recombinant virus can replicate facilitating the rapid production of multiple recombinant viruses on automated platforms in a one-step procedure. Using this vector allowed us to automate the generation of multiple recombinant viruses with a robotic liquid handler and then rapidly screen infected insect cell supernatant for the presence of secreted proteins.
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.
Cell Engineering, 2014
The insect cell-baculovirus protein expression vector system (BEVS) has gained increasing attention as more of its products are approved for human use. However, the system has been relevant for many years, being used for the manufacturing of recombinant veterinary vaccines, as a workhorse in the research laboratory, as an important tool for new drug discovery and as an important source of commercial materials and reagents for research. In this chapter, the key elements that should be considered for the design of a productive BEVS process are discussed, along with a presentation of the state of the art of the system.
Journal of Biotechnology, 2016
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. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of noninfectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts.
Baculovirus-driven protein expression in insect cells: A benchmarking study
Journal of structural biology, 2018
Baculovirus-insect cell expression system has become one of the most widely used eukaryotic expression systems for heterologous protein production in many laboratories. The availability of robust insect cell lines, serum-free media, a range of vectors and commercially-packaged kits have supported the demand for maximizing the exploitation of the baculovirus-insect cell expression system. Naturally, this resulted in varied strategies adopted by different laboratories to optimize protein production. Most laboratories have preference in using either the E. coli transposition-based recombination bacmid technology (e.g. Bac-to-Bac®) or homologous recombination transfection within insect cells (e.g. flashBAC™). Limited data is presented in the literature to benchmark the protocols used for these baculovirus vectors to facilitate the selection of a system for optimal production of target proteins. Taking advantage of the Protein Production and Purification Partnership in Europe (P4EU) scie...
PLoS ONE, 2014
Here we describe the development of a baculovirus vector expression cassette containing rearranged baculovirus-derived genetic regulatory elements. This newly designed expression cassette conferred significant production improvements to the baculovirus expression vector system (BEVS), including prolonged cell integrity after infection, improved protein integrity, and around 4-fold increase in recombinant protein production yields in insect cells with respect to a standard baculovirus vector. The expression cassette consisted of a cDNA encoding for the baculovirus transactivation factors IE1 and IE0, expressed under the control of the polyhedrin promoter, and a homologous repeated transcription enhancer sequence operatively cis-linked to the p10 promoter or to chimeric promoters containing p10. The prolonged cell integrity observed in cells infected by baculoviruses harbouring the novel expression cassette reduced the characteristic proteolysis and aberrant forms frequently found in baculovirus-derived recombinant proteins. The new expression cassette developed here has the potential to significantly improve the productivity of the BEVS.
Opportunities and challenges for the baculovirus expression system
Journal of Invertebrate Pathology, 2011
In this review background information on the baculovirus-insect cell expression system and its applications for producing protein subunits and virus-like particles for vaccine and other purposes is provided. This review will illustrate the principle structure of baculovirus vectors commonly used for heterologous gene expression in insect cells and describe adaptations that have been made over the last 10 years to improve the system in terms of quality of the protein produced and stability of the baculovirus genome. These improvements include enhanced trafficking, folding and glycosylation of the recombinant protein as well as preventing intracellular degradation. Challenges and progress in stabilizing the baculovirus genome in order not to lose the transgene cassette will also be discussed. Recent developments such as how to make multiple alterations in the baculovirus genome without accumulating marker genes are included.