Expression and Interactions of Kinetoplastid Kinetochore Proteins (KKTs) from Trypanosoma brucei.

Abstract

Kinetochores are the macromolecular protein complex that drives chromosome segregation by interacting with centromeric DNA and spindle microtubules in eukaryotes. Kinetochores in well studied eukaryotes bind DNA through widely conserved components like Centromere Protein (CENP)-A and bind microtubules through the Ndc80 complex. However, unconventional type of kinetochore proteins (KKT1-20) were identified in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei (T. brucei), indicating that chromosome segregation is driven by a distinct set of proteins. KKT proteins are comprised of sequential α-helixes that tend to form coiled-coil structures, which will further lead to polymerization and misfolding of proteins, resulting in the formation of inclusion bodies. We expressed and purified the stable KKT proteins with Maltose Binding Protein (MBP) fusion tag in E. coli or Protein A tag in Human Embryonic Kidney (HEK) 293T cells. Furthermore, we identified interactions among KKT proteins using yeast two-hybrid system. The study provides an important basis for further better understanding of the structure and function of KKT proteins.