Purification of Strep-tagged NS1 Protein Complex and Identification of Interacting Proteins

SRECs’ were infected with SIV/SK-544 at an MOI of 2. At 16 h post infection (h.p.i.), cells were harvested in cell lysis buffer (Cell Signaling Technology) with protease inhibitor (Complete protease inhibitor cocktail tablets – Roche Diagnostics Corporation). The lysate was sonicated and then clarified by centrifugation at 16200 × g for 10 min at 4°C. The Strep-tactin sepharose resin (IBA) was washed three times with four column volume (CV) of cell lysis buffer and then was added to the clarified lysate and incubated at 4°C overnight. Next, the lysate-sepharose mixture was added to a polypropylene column (Qiagen) and the sepharose was washed extensively with wash buffer [100 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1 mM EDTA]. The Strep-tag NS1 protein complex was then eluted from the sepharose resin with 3 CV of elution buffer (wash buffer with 2.5 mM desthibiotin). Protease inhibitor cocktail was added to the eluent to prevent degradation of the proteins and the proteins present in the complex were identified by LC-MS/MS at the University of Victoria-Genome BC Proteomics Centre.