Magnetic Resonance Imaging is a noninvasive, complex imaging modality that uses contrast agents to enhance sensitivity and resolution for a clear image enhancement. To optimize the relaxation and targeting properties, our lab has created several protein-based contrast agents, ProCAs. To enable various biophysical characterizations and preclinical studies, we need to optimize expression and purification of the engineered ProCAs. ProCA1 variants are cultured in various cell strains during a tag-less method. ProCA32 with multiple binding sites is also successfully purified. It has a strong metal-binding capability with a relaxivity (r2: 27 mM-1s-1; 0.54; r1: 20.6 mM-1s-1; 0.20 mM-1s-1) that is significantly greater than the clinically used contrast agent,diethylene triamine pentaacidic acid, DTPA (r1: 3.8 mM-1s-1). Our findings in optimizing expression and purification conditions, structural conformation, metal binding, and relaxivity measurements of these ProCAs will facilitate in vitro and in vivo studies needed to move forward into the pre-clinical then clinical phase.