Expression and Purification of Human Senataxin in MCF-7 Cells
Abstract
The human helicase Senataxin is encoded by the SETX gene, and functions in the termination step of transcription, as well as coordinating DNA replication with transcription. Patients with Ataxia oculomotor apraxia type 2 (AOA2) and others with the juvenile onset of amyotrophic lateral sclerosis (ALS4) have mutations in the SETX gene. The mechanism of Senataxin function is poorly understood, and uncovering these mechanisms will yield greater understanding into diseases such as AOA2 and ALS4. Senataxin is the homolog of the yeast protein encoded by the Sen1p gene, which is currently being studied by Dr. Antony's group. Truncated portions of yeast Sen1p have been successfully purified and studied, but without the full human homolog, studies delving into the varying effects of the 40 different mutations seen in AOA2 and ALS4 patients will be limited. My goal will be to create a working human cell line that contains the full SETX gene, and successfully produce the Senataxin protein. I will be adapting a strategy used to purify the large BRCA2 protein and use two maltose binding solubility tags in tandem to purify the Senataxin protein.
Expression and Purification of Human Senataxin in MCF-7 Cells
The human helicase Senataxin is encoded by the SETX gene, and functions in the termination step of transcription, as well as coordinating DNA replication with transcription. Patients with Ataxia oculomotor apraxia type 2 (AOA2) and others with the juvenile onset of amyotrophic lateral sclerosis (ALS4) have mutations in the SETX gene. The mechanism of Senataxin function is poorly understood, and uncovering these mechanisms will yield greater understanding into diseases such as AOA2 and ALS4. Senataxin is the homolog of the yeast protein encoded by the Sen1p gene, which is currently being studied by Dr. Antony's group. Truncated portions of yeast Sen1p have been successfully purified and studied, but without the full human homolog, studies delving into the varying effects of the 40 different mutations seen in AOA2 and ALS4 patients will be limited. My goal will be to create a working human cell line that contains the full SETX gene, and successfully produce the Senataxin protein. I will be adapting a strategy used to purify the large BRCA2 protein and use two maltose binding solubility tags in tandem to purify the Senataxin protein.