Purification and Characterization of Human Leucine-Rich Pentatricopeptide Repeat Containing Protein (LRPPRC) Expressed in Escherichia Coli

Open Access
Gilmore, Andrew Charles
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Craig Eugene Cameron, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
  • Protein
  • Purification
  • French Canadian Leigh Syndrome
  • Escherichia Coli
  • Mitochondria
French-Canadian Leigh Syndrome is caused by mutations in the gene encoding leucine rich pentatricopeptide repeat containing protein (LRPPRC). This disease is characterized by movement disorders (typical of energy deprivation disorders caused by mitochondrial dysfunction) the most common being dystonia. It may also present with seizures, loss of developmental skills, and culminate in kidney failure and heart problems. Consistent with a role in mitochondrial gene expression is the observation that the abundance of the mitochondrial mRNAs for two subunits of cytochrome c oxidase (COX1 and COX3) is reduced in patients with French-Canadian Leigh Syndrome. It is therefore hypothesized that mutations in LRPPRC disrupt the proteins ability to bind and stabilize these mitochondrial mRNAs for proper gene expression. In this thesis, we set out to establish an effective method for purification of LRPPRC. We also investigated the ability of LRPPRC to bind RNA and stimulate mitochondrial transcription in vitro. We succeeded in developing an effective and efficient purification process utilizing the pSUMO system. The purified LRPPRC protein was capable of binding RNA under conditions of low ionic strength. In addition, in vitro mitochondrial transcription assays revealed the potential for LRPPRC to stimulate transcription. Samples that contained purified LRPPRC protein had increased activity as compared with those that lacked LRPRRC. The development of this expression and purification procedure along with the corresponding biochemical assays can now be used to test the hypothesis that mutations in LRPPRC impair its normal function in the mitochondria that is required for proper gene expression.