Viral proteomics: The emerging cutting-edge of virus research

Zhou, ShengTao; Liu, Rui; Zhao, Xia; Huang, CanHua; Wei, YuQuan

While the Y2H method serves as the initial identification of protein-protein interactions, further confirmation of the interaction requires other pull-down assays. These methods involve the expression of one or both of the proteins in another organism, usually E. coli, and the addition of affinity tags such as His6 and GST to the N-or C-terminus of the expressed proteins to improve purification and identification. If only one of the proteins is expressed recombinantly, a native source of the second partner must be available [37] . Several studies have used this method to investigate protein-protein interactions. A recent report unraveled the expression of pituitary tumor-transforming gene 1 (PTTG1)/ securin in hepatitis B virus (HBV)-associated liver diseases using glutathione S-transferase pull-down and co-immunoprecipitation experiments [43] . They concluded that the interaction between PTTG1 and the Skp1-Cul1-F-box ubiquitin ligase complex (SCF) was partially disrupted, possibly through a mechanism involving protein-protein interactions of HBx with PTTG1 and/or SCF. Thus, pull-down experiments can validate Y2H interaction results in virus research. This strategy is potentially useful for the definition of binding partners that conduct signals, as well as for interactions that occur when the host and the virus fuse [26] .