Plants are increasingly used as alternative expression hosts for the production of recombinant proteins offering many advantages including higher biomass and the ability to perform post-translational modifications on complex proteins. Key challenges for optimized accumulation of recombinant proteins in a plant system still remain, including endogenous plant proteolytic activity, which may severely compromise recombinant protein stability. Several strategies have recently been applied to improve protein stability by limiting protease action such as recombinant protein production in various sub-cellular compartments or application of protease inhibitors to limit protease action. A short update on the current strategies applied is provided here, with particular focus on sub-cellular sites previously selected for recombinant protein production and the co-expression of protease inhibitors to limit protease activity.

There is a growing interest in applying tobacco agroinfiltration for recombinant protein production in a plant based system. However, in such a system, the action of proteases might compromise recombinant protein production. Protease sensitivity of model recombinant foot-and-mouth disease (FMD) virus P1-polyprotein (P1) and VP1 (viral capsid protein 1) as well as E. coli glutathione reductase (GOR) were investigated. Recombinant VP1 was more severely degraded when treated with the serine protease trypsin than when treated with the cysteine protease papain. Cathepsin L- and B-like as well as legumain proteolytic activities were elevated in agroinfiltrated tobacco tissues and recombinant VP1 was degraded when incubated with such a protease-containing tobacco extract. In silico analysis revealed potential protease cleavage sites within the P1, VP1 and GOR sequences. The interaction modeling of the single VP1 protein with the proteases papain and trypsin showed greater proximity to proteolytic active sites compared to modeling with the entire P1-polyprotein fusion complex. Several plant transcripts with differential expression were detected 24 hr post-agroinfiltration when the RNA-seq technology was applied to identify changed protease transcripts using the recently available tobacco draft genome. Three candidate genes were identified coding for proteases which included the Responsive-to-Desiccation-21 (RD21) gene and genes for coding vacuolar processing enzymes 1a (NbVPE1a) and 1b (NbVPE1b). The data demonstrates that the tested recombinant proteins are sensitive to protease action and agroinfiltration induces the expression of potential proteases that can compromise recombinant protein production.

Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia) facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin) system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development

The civil unrest that ravaged Angola for nearly 30 years took a heavy toll on the country's wildlife, and led to a lengthy absence of reliable information for many threatened species, including the cheetah Acinonyx jubatus and African wild dog Lycaon pictus. Using camera trapping we assessed the status of these two species in two areas of southern Angola, and complemented our findings by reviewing recent survey reports and observations to provide an update on the species' status. We found unequivocal evidence that African wild dogs are resident and reproducing in Bicuar National Park, where cheetahs appear to be absent. Conversely, cheetahs may be resident in western Cuando Cubango province, where African wild dogs may only be transient. Based on these and other recent records in Angola, we recommend a revision of these species' distribution ranges and note the need for monitoring of these remnant populations and for appropriate attention to any threats.