Rozen-Gagnon Kathryn, Moreland Nicole J, Ruedl Christiane, Vasudevan Subhash G
Program in Emerging Infectious Diseases, DUKE-NUS Graduate Medical School, Singapore.
Protein Expr Purif. 2012 Mar;82(1):20-5. doi: 10.1016/j.pep.2011.11.003. Epub 2011 Nov 11.
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.
登革病毒（DENV）编码的非结构蛋白1（NS1）是一种由352个氨基酸组成的蛋白质，它以多种寡聚状态存在，并且在黄病毒科中具有保守性。尽管NS1因其诊断用途已得到大量研究，但在理解其在一系列病毒过程中的作用方面仍存在差距，这些过程包括复制以及诸如血管渗漏等临床病理的发展。其中许多功能涉及与病毒和宿主蛋白的未知相互作用。本研究描述了一种与DENV1和DENV2的NS1发生反应的小鼠单克隆抗体（mAb 56.2）的产生，以及杆状病毒中重组SUMOstar标签的DENV2 NS1（DENV2 S∗-NS1）的表达。这是首次将登革病毒NS1作为SUMOstar融合蛋白生产，与未标记S∗的NS1构建体相比，S∗标签增加了蛋白的溶解性和分泌性。该蛋白可通过mAb 56.2免疫亲和柱轻松纯化，通过用烟草蚀纹病毒蛋白酶处理去除S∗标签可获得无标签的NS1。尺寸排阻色谱法和糖基化分析表明，分泌的S∗-NS1和切割后的NS1均为六聚体且被糖基化，它们将成为阐明登革病毒NS1蛋白相互作用和功能的有用工具。
