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.
Tag: <span>protein expression</span>
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….
Proteins and their promise for revolutionizing drug discovery have come virtually full circle in just a few decades. The advent of genetic engineering and the emergence of early recombinant proteins such as insulin and interferon dramatically boosted the perceived value of proteins in pharmaceutical research and of protein drugs in particular. Although the lights dimmed somewhat on the promise of therapeutic proteins in subsequent years, more recent times have seen a resurgence of interest in proteins, particularly monoclonal antibodies. Perhaps most telling has been the dawn of the post-genomic era, which has cast a bright spotlight on proteins, long respected as the work-horses of the cell, for their usefulness in exploring cell function, unraveling biochemical pathways, understanding disease, and for their massive value as novel drug targets…
The use of plants as protein expression hosts for human therapeutic proteins is emerging as a safe and cost-effective alternative to microbial and mammalian cell culture. Pharmaceutical protein production is typically carried out in microbes and mammalian cell culture because of their high production potential and/or ability to produce complex eukaryotic proteins. However, immense costs are typically required for production facilities to support their growth. To offset these costs, companies usually build and expand a production facility over several years. In fact, it has been predicted that the demand for high-value pharmaceuticals produced by cell culture will quickly surpass the ability of pharmaceutical companies to produce them…
The first use of mammalian cell culture for the production of vaccines dates back to polio vaccine development in the 1950s. The development of hybridoma technology in the 1970s further stimulated the use of mammalian cells for the production of monoclonal antibodies. Together with developments in genetic engineering, it therefore became possible to produce a wide range of recombinant proteins as well as to improve cell metabolism. Animal cells are now widely used in industrial processes to obtain complex glycoproteins with correct posttranslational modifications and biological activity for diagnostic and therapeutic applications. Animal cells are the main source for commercially available recombinant proteins such as tissue plasminogen activator (tPa), erythropoietin (EPO), DNAse, factor VIII, interferon-ß, and monoclonal antibodies…
At the onset of modern-day biotechnology, products typically fell into two distinct categories, the traditional high volume, low value products (e.g. beer and industrial enzymes) that had come to characterize the biotechnology industry, and low volume, high cost products. Recombinant proteins, the result of technological advances in molecular biology, have come to typify these latter products. Recombinant protein therapeutics have been hugely successful, potentially outstripping production capacity and continue to drive much of the biotechnology. Meanwhile, many recombinant proteins, those characterized as research tools and reagents, are governed by a price-volume relationship typical of industrial enzymes. In a competitive environment, they are fast becoming commodities — price sensitive, packaged as kits, coupled to instrumentation, and relying on heavy marketing and brand recognition. Ominously, the advantage protein therapeutics have enjoyed with patent protection and regulatory constraints on production is being threatened as patents expire and competition from generics increases…