2009)

2009). of IbpA DR2 expression during silicon limitation and growth arrest. These culture conditions eliminate the flux of cellular resources into cell division processes, yet do not limit protein expression. In addition to improving protein expression levels by molecular manipulations, yield was dramatically increased through cultivation enhancement including elevated light and CO2 supplementation. We substantially increased recombinant protein production over starting levels to 1 1.2% of the total sodium dodecyl sulfate-extractable protein in is the most extensively developed system for recombinant protein production in algae. Variability in expression levels has been documented, ranging from a fraction of a percent to 10% of total soluble protein, which relates to the specific protein being expressed (Rasala and Mayfield 2014). It has also been demonstrated that proteins requiring complex folding, such as antibodies, can be produced successfully with this system (Tran et al. UF010 2009) and that cells can be directed to UF010 secrete recombinant proteins into the surrounding medium (Lauersen et al. 2013). Protein expression in is generally limited to the chloroplast because foreign genes introduced into the nucleus are subjected to silencing, and attempts at nuclear-based expression have resulted in low yields (Jones et al. 2012). Further, proteins expressed in the chloroplast lack post-translational modifications due to the bacterial origin of plastids. However, improvements in nuclear transgene stability and protein expression have been reported using a combined production and secretion approach (Lauersen et al. 2013). Until recently, the technology dictated non-photosynthetic growth by addition of UF010 a carbon source (Jones et al. ER81 2012); however, new developments have enabled protein UF010 production under autotrophic conditions (Gimpel et al. 2015). Diatoms are a highly productive class of unicellular eukaryotic microalgae that contribute significantly to the global carbon cycle (Granum et al. 2005). Diatoms generally outcompete other UF010 algae in terms of growth (Brzezinski et al. 2001; Carter et al. 2005), and their high productivity indicates an intrinsic efficiency in converting sunlight and CO2 to useful products. For example, diatoms are promising candidates for algal-based biofuel production, in which low cost and high productivity are essential (Sheehan?et al. 1998; Hildebrand et al. 2012). Diatoms have unique features, including cell walls (referred to as frustules) which are comprised of nanostructured silica. More specifically, the frustule is composed of structures that consist of an amalgam of mesoporous particulate silica 10C100?nm in size (Chiappino and Volcani 1977; Schmid and Schulz 1979; Crawford et al. 2001; Noll et al. 2002). Although it presents a barrier, the cell wall is easily disrupted by detergent treatment or sonication, the former of which disrupts connections between larger silica cell wall structures and the latter of which generates small silica particles. Interestingly, nanoparticulate mesoporous silica has been shown to have efficacy as an adjuvant by boosting the immune response (Carvalho et al. 2010; Mody et al. 2013). We recently demonstrated the adjuvanticity of diatom silica (diatomaceous earth) in an immunization study with Newcastle disease virus in chickens (Nazmi et al. 2016). A consequence of frustule formation in diatoms is a strict requirement for silicon, the availability of which controls cell cycle progression. Favorable metabolic conditions, related to the availability of carbon, nitrogen, and cellular energy that can be channeled into protein synthesis rather than biomass accumulation, occur during silicon limitation (Darley and Volcani 1969). Silicon metabolism is generally uncoupled from nitrogen metabolism (Claquin et al. 2002); thus, silicon starvation would be expected to have little effect on protein synthesis potential. In the most highly developed recombinant protein expression systems, such as bacteria and yeasts, conditions in which cellular growth is blocked or retarded are desirable because they permit surplus energy and severely.