UT Chem/Biochem Dept

The 2004-2005 Beckman Scholars: Sarah Rodriguez

Beckman research project in the Iverson group:

Chemistry of Antibody Binding Affinity and Specificity Towards Bacillus anthracis Toxin

Antibodies have proven to recognize target molecules with considerable affinity and specificity. This property of antibodies has led to the field of antibody engineering targeted toward a specific molecule for diagnostic and therapeutic purposes. The construction of a high affinity and specificity antibody that effectively neutralizes the protective antigen fragment of the Bacillus anthracis toxin is a major aim of the labor. The Bacillus anthracis toxin is composed of three subunits: lethal factor, responsible for destruction of cells of the immune system, edema factor responsible for tissue damage with the release of fluid into the lungs, and the protective antigen which binds to cell surfaces and forms a channel in the membrane which allows for the entry of the previously described two subunits. Neutralization of the protective antigen with the binding of a properly engineered antibody aims to prevent the major cellular destruction caused by the lethal factor and edema factor of the Bacillus anthracis toxin.

Specifically, I currently work on a project that aims to obtain structural data of the antibody which expresses the highest affinity for protective antigen using X-ray crystallography. Because it is the variable region of the antibody which is responsible for the binding affinity and neutralization of the protective antigen subunit, only this fragment of the antibody will be crystallized. E. Coli is used for antibody production to express the single chain variable fragment within the periplasmic space of the cell. Cells are then subject to osmotic shock using lysosyme EDTA for destruction of the outer cellular membranes. Because the variable fragment is produced with a histadine tag, it can be purified using a Ni NTA column. This is then followed by the further purification using fast performance liquid chromatography (FPLC). The entirety of the antibody fragment production procedure used to prepare the antibody fragment is optimized to obtain the largest amount (mg) of protein possible, which is then concentrated. The protein undergoes various crystallization conditions to find the optimal environment specific to the antibody fragment for its crystallization. Once formed, the antibody fragment crystal will be interpreted using X-ray diffraction. The solved crystal structure will provide valuable information with which to interpret the underlying causes for molecular binding affinity and neutralization.

Download a copy of Sarah's paper on Evolving Affinity Enhanced Antibodies: High Mutational Rates for Discovery of Coupled Mutations .