SUMOylation, the covalent attachment of SUMO (small ubiquitin-like modifier), is a eukaryotic posttranslational event that has been demonstrated to play a critical role in several biological processes. When used as an N-terminal tag or fusion partner, SUMO has been shown to enhance functional protein production significantly by improving folding, solubility, and stability. We have engineered several SUMOs and, through their fusion, developed a system for enhancing the expression and secretion of complex proteins. To demonstrate the fidelity of this fusion technology, secreted phospholipase A 2 proteins (sPLA 2 ) were produced using HEK-293T and CHO-K1 cells. Five mouse sPLA 2 homologs were expressed and secreted in mammalian cell cultures using SUMO or SUMO-derived, N-terminal fusion partners. Mean and median increases of 43-and 18-fold, respectively, were obtained using novel SUMO mutants that are resistant to digestion by endogenous deSUMOylases.Keywords: SUMO; SUMO fusion; phospholipase A 2 ; mammalian expression; deSUMOylase SUMO (Small Ubiquitin-like MOdifier) and the SUMO pathway are highly conserved throughout eukaryotes. SUMOylation of proteins in humans was discovered recently by Blobel and colleagues in studies of protein compartmentalization (Matunis et al. 1996). Although SUMO has low (18%) sequence identity with ubiquitin, the two proteins are very similar structurally. The SUMO and ubiquitin pathways also share many features, for instance, both proteins are synthesized as precursors requiring a proteolytic processing step prior to target protein conjugation, and each has a series of activating, conjugating, and ligating enzymes that attach the C termini of the respective proteins to the e-amino group of lysines in the target proteins (Johnson 2004). SUMO and ubiquitin hydrolases both play important regulatory roles by their removal of SUMO or ubiquitin from target proteins; however, the mechanistic similarity ends at the fate of the modified protein. Unlike ubiquitin, SUMO is not a signal for proteolysis by the 26S proteasome. Although SUMOylation is recognized as an important regulatory modification, its precise role is still the subject of active study. Several papers implicate SUMOylation as a translocation signal for nuclear-cytosolic trafficking. Paradoxically, lysine residues in target proteins that are SUMOylated are protected from ubiquitylation, while SUMOylation of proteins can also serve as a signal for ubiquitylation (Prudden et al. 2007). Thus, nature has evolved two pathways that have remarkably similar mechanical and biochemical features, yet have very distinct roles in cellular physiology (Johnson 2004).Fusion to SUMO has been shown to enhance the expression of recombinant proteins in Escherichia coli (Malakhov et al. 2004;Butt et al. 2005;Zuo et al. 2005;Marblestone et al. 2006;Panavas et al. 2008), while fusion to hSUMO3 led to similar enhancement in E. coli (data not shown) and Pichia pastoris (R.J. Peroutka, D. Roy, J. Strickler, and T.R. Butt, in prep.). Based on the or...

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This is a case of coccidioidomycosis in a dog, examined for vomiting and labored breathing. Physical examination and thoracic and abdominal imaging revealed pleural and peritoneal effusions, both of which exhibited neutrophilic inflammation with a substantial eosinophilic component. The dog had positive IgM and IgG coccidioidomycosis titers at initial evaluation. The eosinophilic component of the inflammation was attributed to coccidioidomycosis. The dog underwent approximately 6 months of fluconazole treatment, with both effusions and clinical signs improving after 6 weeks. Three months after cessation of antifungal treatment, the dog developed a mid‐diaphyseal lytic and proliferative lesion in the left radius caused by Coccidioides spp. This case illustrates the importance of consideration of coccidioidomycosis when an eosinophilic cavitary effusion is present in dogs that live in or have traveled to endemic regions.