Expression and Purification of nsP2 Protease of Chikungunya Virus.

The nonstructural protein 2 (nsP2) of chikungunya virus (CHIKV) is an essential cysteine protease involved in viral polyprotein processing and replication complex regulation. Biochemical and structural studies of nsP2 require the availability of highly pure and enzymatically active protein. In this chapter, we describe a straightforward protocol for the heterologous expression and purification of CHIKV nsP2 protease domain (nsP2pro) in Escherichia coli. The protein is expressed at low temperature as a His-tagged construct and purified under native conditions by immobilized metal ion affinity chromatography, followed by dialysis for buffer exchange. The procedure typically yields milligram quantities of highly pure (>95%) and active nsP2pro, and has been successfully applied to wild-type and mutant variants for enzymatic assays and inhibitor screening.