Furthermore, by lowering the expression of the E1 and E2, additional cellular resources can be Bortezomib diverted towards production of the target protein. Even without any process optimization, our E3-dependent SUMO conjugation system yielded,5 mg/L of mono-sumoylated protein. Second, the system enables functional characterization of any of the sumoylation cascade enzymes while eliminating the concern for localization, downstream interactions, and the diversity of sumoylated proteins that can obscure similar analysis in eukaryotic hosts. Our system also produces physiologically relevant results. For instance, we observed that Smad4 was sumoylated primarily at K159, which is reported to be the major sumoylation site. We did not detect sumoylation at position K113, which was reported as a minor site of sumoylation in one report but was not sumoylated in another. We also did not detect SUMO-1 chains on target proteins in our E3-dependent system, which is in stark contrast to an earlier bacterial E3-independent sumoylation system. It should be noted, however, that the inability of MS analysis to reveal poly-sumoylation via K16 and K17 linkages on SUMO-1 could arise from low abundance and/or poor ionization efficiency of these species. Nonetheless, based on the high-intensity MS signal detected for the K159 SUMO-1 peptide, we conclude that no appreciable quantities of SUMO-1 chains are present. Overall, our system yields results that are entirely consistent with the known molecular biology of sumoylation. As a corollary, we show that engineered E3 variants can be expressed and functionally characterized in our system. This is significant because our bacterial SUMO-conjugation system provides a potentially less convoluted background for studying sumoylation. While in vitro reconstitution studies could also be used to eliminate these factors, our system obviates the need for purification of each cascade component and the corresponding need to modify each cascade component with a purification tag, which can affect enzyme function. Thus, we anticipate that our sumo-engineered E. coli system will be a useful new tool for illuminating the molecular details of the SUMO-conjugation process. Of the numerous peptides generated though sequential complement cleavage, the anaphylatoxins, C3a and C5a, are among the most potent of all known inflammatory mediators. By binding to their cognate receptors, the C3a receptor and C5a receptor, these peptides mediate their inflammatory effects across a variety of pathologic settings by promoting vascular permeability, leukocyte activation, and chemotaxis. Modulation of complement in animal models of stroke has proven effective in suppressing post-ischemic inflammation. Recently, a role for complement activation in tissue regeneration has been proposed. More specifically, complement may regulate the process of endogenous neurogenesis, as neural progenitor cells and immature neurons have been reported to express both C3aR and C5aR.