Expression and purification of INI1 truncations

Abstract

INI1 (Integrase Interactor 1) is a highly-conserved, core component of the SWI/SNF remodeling complex and is characterized as a tumor suppressor. Mutations in the SMARCB1 gene/INI1 protein cause aggressive pediatric cancers called malignant rhabdoid tumors (MRT). Atypical Teratoid/Rhabdoid Tumors (AT/RT), a common form of MRT, are a pediatric cancer associated with INI1 mutations and form in the central nervous system and kidneys of infants. Although the tertiary structure of INI1 is unknown, the 385 amino acids-long protein contains an N-terminal DNA binding domain (1-186), two imperfect repeat regions (187-245 and 259-319) that could mediate protein-protein interactions, and a C-terminal coiled-coil domain (334-376). Little is understood about INI1's function, structure, regulation, or DNA binding. To determine INI1's minimal binding domain, INI1(31-125) and INI1(31-144) were expressed in E. coli cells. The 6X His-tagged truncations did not bind to the Ni-NTA resin, necessitating the use of binding tests. The results of this study conclude that the INI1 truncations did not bind to the Ni-NTA resin under any tested conditions, indicating that further attempts need to be made at finding the correct binding condition and ensuring that a functional 6X His-tag is present. In future studies, the purified INI1(31-125) and INI1(31-144) fragments will be used in DNA binding assays to determine if these fragments contain INI's minimum binding domain.