Expression and Purification of the HBO1/JADE1 Histone Acetyltransferase Complex

Open Access
Author:
Tollemar, Viktor Clifford
Area of Honors:
Biochemistry and Molecular Biology
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Song Tan, Thesis Supervisor
  • David Scott Gilmour, Honors Advisor
  • Scott Brian Selleck, Faculty Reader
Keywords:
  • protein
  • expression
  • complex
  • hbo1
  • jade1
  • biochemistry
  • molecular biology
  • nucleosome
Abstract:
Normal human development and cell function require the regulation of gene expression. Our genetic material is packaged as chromatin and this packaging directly affects how genes are regulated. The repeating unit of chromatin, the nucleosome, consists of DNA wrapped around an octamer of histone proteins. An important field of epigenetic research focuses on chromatin enzymes that regulate gene expression by chemically modifying histone proteins in a nucleosome. Specifically, the histone acetyltransferase (HAT) class of enzymes acetylate histone tails, a chemical modification that is closely associated with gene activation. The histone acetyltransferase binding to ORC (HBO1) HAT enzyme is of particular interest to me. HBO1 binds to the origin of replication complex (ORC), which is critical to the initiation and regulation of DNA replication. The exact relationship between acetylation and initiation of DNA replication remains unclear, but it is known that HBO1’s HAT enzymatic activity is significant in DNA replication. It is also known that HBO1 requires a protein partner called JADE1 in order to effectively carry out its catalytic function on the nucleosome. How the HBO1/JADE1 complex interacts with the nucleosome is currently under investigation. The ultimate goal of my project is to determine the structure of the HBO1/JADE1 complex bound to the nucleosome in order to elucidate the specific physical interactions between complex and nucleosome. To that end, I have created a polycistronic expression vector containing both genes, I have co-expressed the HBO1/JADE1 complex, and I have attempted to purify the complex using metal affinity chromatography.