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>recombinant baculovirus</span>
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 Typically, in most baculovirus cloning vectors the coding region of the polyhedrin gene has been replaced by a polylinker with multiple cloning sites for the insertion of the cDNA of interest downstream of the strong polyhedrin promoter…
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….
Parvoviruses are one of the most prevalent infectious agents in the laboratory rodent. Their effect on research can range from immune dysfunction that may mislead researchers when interpreting results to lethal effects on animals. Until recently parvovirus infection in mice was thought to be caused by minute mouse virus (MMV) and in rats by rat viral agents in the KRV or H-1 serogroups. Relatively newly discovered viruses in these groups are mouse (MPV) and rat parvoviruses (RPV-1 and 2). Parvoviruses are 15–20 nm in diameter and are single-stranded DNA viruses of about 5,000 nucleotides, which replicate through a double-stranded DNA intermediate. The protein composition consists of three structural or capsid proteins providing the viral coat (VP-1, VP-2, and VP-3) and two non-structural proteins involved in viral replication (NS-1 and NS-2). Among the capsid proteins,VP-2 is the major protein…
G protein-coupled receptors (GPCRs) comprise a “superfamily” of cell surface receptors that play a prominent role in cell signalling and are classified into more than 100 subfamilies according to sequence, ligand structure, and receptor function. They are cell surface receptor proteins with seven transmembrane domains which transduce extracellular signals to the interior of cells through heterotrimeric G proteins. GPCRs’ exposure at the exterior cell surface and strong role in cell regulation has provided a rich target family for small compound therapeutics. Of the estimated 35,000 genes in the human genome, approximately 750 encode for GPCRs; half likely encoding sensory receptors, the remaining half representing potential drug targets. Only about 30 of these potential targets are currently modulated by existing pharmaceuticals with approximately 400 remaining potential pharmaceutical targets for validation…