Dengue virus (DENV) encoded nonstructural one (NS1) is a 352 amino acid protein that exists in multiple oligomeric states and is conserved within the flavivirus family. Although NS1 has been heavily researched for its diagnostic utility, there is a gap in the understanding of its role in a range of viral processes, including replication and development of clinical pathologies such as vascular leakage. Many of these functions involve unknown interactions with viral and host proteins. This study describes the generation of a mouse monoclonal antibody (mAb 56.2) that reacts with NS1 from DENV1 and 2, and the expression of recombinant SUMOstar-tagged DENV2 NS1 (DENV2 S*-NS1) in baculovirus. This is the first time dengue NS1 has been produced as a SUMOstar fusion with the S*-tag increasing protein solubility and secretion compared with a non-S*-tagged NS1 construct. The protein was readily purified using a mAb 56.2 immunoaffinity column and untagged NS1 was obtained by treatment with tobacco etch virus protease to remove the S*-tag. Size exclusion chromatography and glycosylation assays showed that both secreted S*-NS1, and cleaved NS1, are hexameric and glycosylated, and will be useful tools in elucidating dengue NS1 protein interactions and functions. (C) 2011 Elsevier Inc. All rights reserved.