Production of Apolipoprotein B Fragments for Structural Analysis

Regulation of the secretion of hepatic very low density lipoprotein (VLDL) can modulate the plasma level of atherogenic apolipoprotein (apo) B-containing lipoproteins. We have shown that sequences within the ?1 domain of apoB are important for lipid recruitment during assembly, irreversible anchoring of apoB to lipoprotein, and degradation when VLDL assembly is inefficient. We hypothesize that specific apoB polypeptide sequences, with unique structural features, mediate the interaction with hepatocellular components of degradation pathways and that this surveillance system may be compromised with disease or aging leading to enhanced VLDL production.

In this summer research project, we will use bacterial and eukaryotic cell systems to express and purify apoB fragments from the ?2 and ?2 domains of the protein for subsequent characterization of their structural and lipid-binding properties. Fragments of approximately 20 kDa, derived from the ?1, ?2 and ?2 regions of apoB100 will be expressed as hexahistidine-tagged fusion proteins and purified from E. coli or from stably transfected HEK293 cells. The purity of the proteins will be assessed by SDS-PAGE and the identity of each protein region will be verified by immunoblot analysis using a panel of monoclonal antibodies. Preliminary structural analysis will use circular dichroism and the ability of the proteins to bind to phospholipid emulsions and triglyceride droplets will be assessed in vitro. The studies outlined will provide important information on the structural properties of apoB sequences that lead to pre-secretory degradation of apoB.

Faculty Supervisor:

Roger McLeod

Student:

Christian Flores Diaz

Partner:

Discipline:

Biochemistry / Molecular biology

Sector:

University:

Dalhousie University

Program:

Globalink