This project focuses on the development of highly effective non-chromatographic methods for purifying recombinant proteins and enzymes. This report covers work at Ohio State University, which focuses on the further optimization of the purification technology in the PI s new laboratory. A key aspect of the technology involves the use of a self-cleaving protein module, which can be used to make any purification tag self-cleaving. By combining this module with a variety of purification tags, we have generated a number of simple, inexpensive and highly effective methods for protein purification. We have applied these methods to the production of several proteins, including organophosphohydrolase (OPH), which can be used to degrade nerve agents and environmentally persistent insecticides. We have further demonstrated these methods in high cell-density fermentation at laboratory scale, and have provided evidence of their effectiveness. Our most recent work has been on the optimization of the fermentation process itself, as well as a more biochemical optimization of the expression system. Overall, the ARO support on this project has yielded two patent applications, seven peer-reviewed papers and one book chapter. In addition to these, three manuscripts are in late stages of preparation.