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)…
Category: <span>Baculovirus Expression Technology</span>
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…
Baculovirus, particularly AcMNPV (Autographa californica multiple nucleocapsids polyhedrosis virus), is widely used for heterologous protein expression. There are several shortcomings in the current practice of preserving and scaling up baculovirus: 1) extracellular baculovirus stocks, routinely prepared in large volumes and stored at 4º C, are often unstable; 2) laborious and time-consuming steps to amplify and titer the baculovirus stocks are often necessary, and generally recommended, for achieving consistent viral infection and protein expression; 3) once prepared, the baculovirus is suspended and stored in conditioned medium. Given the complex, undefined, and unstable nature of the spent media components, including proteases and nucleases, protein expression tends to vary even when steps are taken to titer the virus stock and adjust the amount of stock used for infection. Here, we will report a new method for preserving and scaling up baculoviruses that: 1) provides a new form of viral stock more stable than the traditional, extracellular stock; 2) eliminates the need for virus amplification and retitering; 3) drastically reduces the turn-around time and resources required for scale-up; and 4) improves yield and consistency in protein expression.
Baculovirus infection of insect cells is an established method to obtain large quantities of biologically active recombinant proteins with properties similar to those of proteins expressed in mammalian cells. Insect cells are capable of most mammalian posttranslational modifications that control protein compartmentalization, secretion, targeting to nucleus or cell surface, N- and O-glycosylation, phosphorylation, proteolytic processing, and assembly of multi-protein complexes. The baculovirus transfer plasmids and accessory products utilized for protein expression in insect cells are currently available from several commercial sources. The plasmids often contain his-tags to facilitate Ni-NTA purification of recombinant proteins, and/or signal peptides to promote or enhance secretion of the proteins into the medium. Typically, in most baculovirus cloning vectors the coding region of the polyhedrin gene has been replaced by a polylinker with multiple cloning sites for the insertion of the cDNA of interest downstream of the strong polyhedrin promoter…
Baculoviruses have found many uses in the field of biology, including as a control strategy for major insect pests such as the gypsy moth, the corn earworm, and the velvetbean caterpillar, for high level expression of recombinant proteins in insect cells, and, more recently, as gene delivery vehicles into mammalian cells. Some of these uses entail the need for isolation of baculoviral DNA for molecular biology studies. Currently, there are three ways in which baculoviral DNA can be obtained: 1) from the infection of insect larvae or cell cultures and the recovery of occlusion bodies (OB) containing viral particles, from which the DNA is then extracted; 2) from the recovery of extracellular virus (ECV) or budded virus (BV), usually from infected cell cultures; or 3) from total intracellular DNA harvested from infected cell cultures early in the infection cycle…
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…