Expression and Purification of the Tumor Suppressor INI1

Abstract

The absence or mutation of a chromatin remodeling protein called INI1, part of the SWI/SNF complex, is linked to the formation of a pediatric cancer called Atypical Teratoid Rhabdoid tumors (AT/RT) which produces tumors on the brain and spinal cord of infants. Due to the low survival rate of this condition and the lack of research on INI1, researchers want to learn all that they can about this protein in hopes to find a cure. We intend to determine the structure of INI1 bound to chromatin using X-ray crystallography and determining if and where INI1 binds to DNA. In preparation for this work, I have expressed and isolated multiple batches of full length INI1 along with five other truncations (1-186, 186-385, 1-104, 259-385, 31-186). To express and isolate the protein and protein fragments, I used heat shock transformation, inoculation, induction, sonication, Ni-resin affinity chromatography, and elution and then visualized the resulting product using SDS-PAGE gel electrophoresis. All of the protein and protein fragments are now at least 50% isolated. Future projects will include further isolating the samples, determining the domains of INI1 which bind to chromatin using gel shift assay, and determining INI1’s structure using X-ray crystallography.