Variation of viral titre and recombinant product yields reported for the baculovirus expression vector system have been attributed to many specific infection variables. These include multiplicity of infection (MOI) and cell density at time of infection and time of harvest, as well as virus bank quality and efficiency. The MOI is defined as the number of plaque forming units (pfu) per cell that are added at the time of infection. Virus titre (pfu/ml) is determined by the plaque assay method. The MOI parameter is easily manipulated and may be important in optimising recombinant protein yields. Other sources of variation during both cell growth and viral infection phases may be responsible for the range of reported yields. Past studies in our laboratory compared the behaviour of cells infected with high and low MOI values, specifically regarding nutrient limitation and deprivation. In addition to these aspects, the quality of the virus bank may be an important factor which influences heterologous protein yields in the insect cell baculovirus system. Thus, production yields may be correlated to virus efficiency…
Tag: <span>BEVS</span>
In general, the industry has gone through another of its realignment periods, where much was learned, but a lot of restructuring and refocusing took place. Driven by the need to keep the doors open, small to medium sized firms had to do some severe belt tightening, or completely redefine themselves as to technologies, products, and personnel. Many of the larger firms reevaluated their product pipelines, and then made the changes they felt were necessary to assure future revenues, or to make themselves attractive merger partners. Numerous large mergers took place with some that were the largest the biopharmaceutical industry has ever seen. In addition, several medium-sized companies merged, or otherwise found strategic alliances that energized their product pipelines, or simply provided the cash they needed to keep going. Antibody products did very well with a number of blockbusters receiving license approval in 2003…
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…
The Sf-9 insect cell/baculovirus expression system is one of the most commonly used protein expression systems. It is the preferred system for generating large amounts of protein in a short period of time, and it has been successfully used to express several hundreds of different proteins. A representative list of the different proteins made in our laboratory over the past decade with the Sf-9 insect cell/BEVS system is given in Table 1. These proteins are often used in drug screening studies and structure function analysis. Proteins intended for therapeutic purposes are not normally produced using this technology, although a few examples do exist. There is also an unexplored potential for the cells to be used for the production of recombinant viral vectors. Recent reports demonstrating the ability of baculoviruses to express proteins in mammalian cells, with mammalian promoters, indicate that BEVS technology might soon have a major role to play in the field of gene delivery…