Optimization of Expression and Purification Methods for the Study of Protein-Based Magnetic Resonance Imaging Contrast Agents

Magnetic Resonance Imaging instruments rely on a contrast agent to provide high-resolution images of tissues in vivo. However, current clinical contrast agents are hindered by low relaxivity and fast correlation time, necessitating high injection dosages. These concerns, among others, have driven the development of a class of protein-based contrast agents (ProCAs), by design of lanthanide binding sites into a scaffold protein. ProCA1 has a higher reported relaxivity and dosage efficiency than current contrast agents. In this study, expression and Glutathione-STransferase purification procedures were optimized, and a refolding method for rapid production of ProCA1 has been developed to enable studies of conformation, metal binding, relaxivity, and in vivo applications. Several ProCA1 variants with 4-5 charged ligand residues were shown to have strong gadolinium binding affinity (Kd of 10 -12 M) and metal selectivity. Several options to improve ProCA1 have been explored, including addition of a polyethylene chain or a bombesin tag. INDEX WORDS: Magnetic Resonance Imaging, Contrast agents, Relaxivity, Gadolinium, GST, Refolding method, PEGylation. OPTIMIZATION OF EXPRESSION AND PURIFICATION METHODS FOR THE STUDY OF PROTEIN-BASED MAGNETIC RESONANCE IMAGING CONTRAST AGENTS