The aim of the research proposed is to investigate the use of new affinity matrices in purification and cleavage of recombinant proteins at process scale. The performance of different affinity matrices for immobilization of enzymes will be directly compared. Scalability and performance will be evaluated in relation to cost. In particular, the performance of membrane- immobilized enzyme will be compared to soft gel and rigid bead immobilized enzyme. The results could aid in design of affinity matrices useful for process scale work. By streamlining purification processes significantly, applicable affinity methods could facilitate the development of new protein therapeutics. A small domain that can be purified by a scalable affinity chromatography method will be defined by peptide synthesis. Peptide sequences taken from calcium-binding domains of proteins such as troponin C or calmodulin will be synthesized. After initial screening to identify peptides having affinity for Ca2+, an affinity purification method based upon metal binding will be optimized. The ultimate goal is to express the shortest active sequence recombinantly in fusion with potential product protein sequences.
