Identification of Human Protein Interaction Domains using an ORFeome-based Yeast Two-hybrid Fragment Library

Physical
interactions between proteins are essential for biological
processes. Hence, there have been major efforts to elucidate the complete
networks of protein–protein interactions, or “interactomes”,
of various organisms. Detailed descriptions of protein interaction
networks should include information on the discrete domains that mediate
these interactions, yet most large-scale efforts model interactions
between whole proteins only. We previously developed a yeast two-hybrid-based
strategy to systematically map interaction domains and generated a
domain-based interactome network for 750 proteins involved in C. elegans early embryonic development. Here, we expand
the concept of Y2H-based interaction domain mapping to the genome-wide
level. We generated a human fragment library by randomly fragmenting
the full-length open reading frames (ORFs) present in the human ORFeome
collection. Screens using several proteins required for cell division
or polarity establishment as baits demonstrate the ability to accurately
identify interaction domains for human proteins using this approach,
while the experimental quality of the Y2H data was independently verified
in coaffinity purification assays. The library generation strategy
can easily be adapted to generate libraries from full-length ORF collections
of other organisms.