Expression, purification, and functional characterization of West Nile Virus NS1 in E. coli host expression system

West Nile Virus (WNV), a member of the Flaviviridae family, is a neurotropic virus that can lead to mild to severe illness. Diagnosing and treating WNV infection is complicated due to cross-reactivity of non-neutralizing antibodies generated after infection with other flaviviruses, also known as antibody-dependent enhancement (ADE). The WNV non-structural protein 1 (NS1) can be localized intracellularly, associated with the membrane, or secreted, making it a promising target for diagnostic and therapeutic applications. Structural and functional studies require scalable and efficient expression of the WNV NS1 protein in its native conformation. However, expressing WNV NS1 in bacterial systems results in a misfolded form of the protein in inclusion bodies, making it unsuitable for further diagnostic or therapeutic studies. Here, we present a detailed protocol for expressing and purifying natively folded WNV NS1 protein in a scalable bacterial expression system that yields ∼8 mg per liter of culture. The refolded NS1 antigen binds effectively to polyclonal antibodies from WNV-infected serum samples, confirming the native antigenic properties of WNV NS1. Our study provides valuable insights into the expression, purification, and design strategies for NS1 antigen-based diagnostics and subunit vaccine candidates against WNV.