The baculovirus expression vector system (BEVS) has emerged as a powerful tool for the production of recombinant proteins used as therapeutics, reagents, and diagnostics. In order to maximize the system’s efficiency and thereby reduce costs, optimizing production parameters is imperative. A critical factor in optimization is the production of a high-quality baculovirus stock with a high-titer, pure clonal population of recombinant virus that is stable over time. Baculovirus stocks may contain alternate varieties of infectious virus due to cross-contamination, outgrowth of non-recombinant virus, and excision of inserts attributable to some recombinant virus production technologies. Since the advent of the BEVS, the “gold standard” for production of pure baculovirus stocks has been plaque purification. Briefly, plaque purification involves infecting a monolayer of cells with dilutions of virus before applying an agarose overlay to the monolayer. After a 5–7 day incubation, isolated plaques can be picked, virus eluted from the agarose plug, and amplified. The drawbacks of plaque purification are: (1) it is time and labor-intensive; (2) the results hinge greatly on the health of the cells and the cell density at infection; (3) identification and picking of isolated plaques is challenging; and (4) the integrity of the procedure is easily compromised by virus diffusion and mass flow of virus-containing liquid beneath the agarose overlay…
Tag: <span>BEVS</span>
Baculoviruses are large rod-shaped DNA-viruses which specifically infect only arthropods, mostly lepidopteran species. Among the over 600 known species, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most characterized and widely used. The baculovirus genome is poorly transcribed and does not replicate in non-target cells. Thus, baculoviruses are harmless for vertebrates even though they can be found everywhere and we eat them daily on our vegetables. Baculoviruses have been applied in biotechnology for almost a century now, first as biopesticides (since the 1920s), then for recombinant protein production (since the early 1980s), and most recently for gene delivery into vertebrate cells (since the mid-1990s)…
ImmBio’s lead development candidate is an influenza vaccine based on the ImmunoBody® platform technology. An ImmunoBody is a fusion of a selected immuno-dominant antigen with a cell-binding domain — the Fc fragment of human IgG1. The use of recombinant Fc fusion proteins is well documented where it can help solubilize hydrophobic proteins, provide a handle for easy detection and purification, as well as improve half-life…
The biologics market, although difficult to estimate, is currently thought to be in excess of $20 billion. In recent years, the growth in the novel therapeutics market has continued to exceed all but the most optimistic of expectations. The number of products in early stage trials may already be over 1,000, with an estimated 40 or so additional products in the process of finally being released to the market. The biologics market is led by relatively few “blockbuster” drugs, but the breadth of novel products continues to expand. This has resulted in exciting times for clinicians but has resulted in concern related to the bottleneck of production capacities for these drugs, as well as the pressure from healthcare agencies to reduce the cost of goods…
The developing biotechnology community may offer solutions and hope for recent world events that have focused attention on the vulnerability of the world’s population. Concerns about new pandemics have been raised by the emergence of new influenza strains and the re-emergence of older and even more highly virulent strains. In addition, there are fears that bioterrorism could involve agents such as anthrax or smallpox, and these threats become even more of a concern when you consider the increased mobility of such organisms via today’s commercial aviation. The ability of the biomedical community to respond rapidly to these shifting threats is more important than ever…
The baculovirus expression vector system, which is based on infecting insect cells with recombinant Autographa californica nuclear polyhedrosis virus (AcNPV), is one of the most commonly used eukaryotic expression systems aimed at producing functionally active mammalian proteins. It offers advantages such as high-level protein expression and post-translational processing capabilities that are extremely important to the biological activity of certain proteins. This system utilizes a strong promoter of the very late gene, polyhedrin, to drive heterologous protein overexpression. Nevertheless, in order to generate milligram amounts of recombinant proteins, cell culture often needs to be scaled up to as much as 25 liters….
The Baculovirus Expression Vector System (BEVS) is widely used for the production of a broad variety of heterologous proteins that are often secreted into the culture medium as soluble, biologically active, properly glycosylated, and correctly folded. Downstream purification of a secreted protein is considerably easier due to the absence of many contaminating cellular proteins and nucleic acids in the culture supernatant. The BEVS system has also successfully been used for the production of virus-like particles (VLPs) for a broad variety of proteins derived from many different viruses…