Figure 2.

S. Typhimurium SspH2 protein complex purification using Virotrap. In Virotrap, a fusion of the bait, e.g., effector protein of interest, to the C-terminus of the Gag polyprotein (myristoylated; pink zigzag line) of the human immunodeficiency virus type 1 (HIV-1) is transiently expressed in HEK293T cells. Along with the Gag-bait fusion, FLAG-tagged vesicular stomatitis virus glycoprotein (VSV-G) is expressed and becomes embedded in the plasma membrane. Gag facilitates multimerization and localization of the fusion protein at the plasma membrane, giving rise to virus-like particle (VLP) budding. Consequently, host preys are trapped inside VLPs that can be captured in the cell medium by anti-FLAG immunoprecipitation. LC-MS/MS-based analysis of the VLP content subsequently allows identification of significant bait co-enriched host proteins.