This SBIR Phase II research develops methods to improve the manufacture of recombinant protein products produced in foreign hosts. Cost-effective production of proteins generally utilizes organisms that are well-suited for protein engineering and large-scale production. Establishing a suitable production system for a protein is often a time-consuming, trial-and-error-based process and can be a significant barrier for the commercialization of a protein. In cases where production systems are found, they are often far from optimized due to the time and cost required as well as our current limited understanding of the critical parameters. In Phase I several gene design variables were assessed for their importance to protein expression in the bacterium Escherichia coli, a commonly used production organism. Data suggested novel means for gene optimization that were unexpected from conventional wisdom. In Phase II relevant gene design variables suggested by Phase I will be explored toward development of a refined model of the relationship of gene design to protein expression in E. coli as well as in other useful production organisms.<br/><br/>The broader impacts of this research are improved manufacturing techniques for recombinant protein based products. Protein products constitute a currently >$40 billion and rapidly growing world-wide market including industrial enzymes, diagnostic enzymes and protein pharmaceuticals. The tools developed from this project will drastically improve the speed, reduce the cost, and remove the uncertainties of modern protein manufacturing, which significantly limit this market. Improved production will also accelerate the study of proteins with therapeutic or otherwise marketable potential, expanding the field of candidate proteins for commercialization.