Development of an optimized feeding technology for dairy cows: Improvement in resistance to ruminal proteases in the de novo-designed protein MB-1

We have previously reported on MB-1, a designer protein with potential application in animal nutrition. Having a high content of selected essential amino acids, MB-1 should provide limiting nutrients for animals and promote growth and production. However, the protein was found to have marginal conformational and proteolytic stability, and, thus, strategies for stabilizing MB-1 were elaborated. We discuss the synthesis of MB-1-Cys dimer, a protein with an intermolecular disulfide bridge. This mutant was exposed to Pronase E protease preparation as well as to proteases extracted from ruminal microbes. It was found that in both cases, MB-1-Cys dimer had a better resistance to proteolytic degradation than MB-1. Denaturation and hydrophobic dye binding studies revealed that this enhanced stability was not owing to conformational stabilization, but rather to changes in surface exposure as a consequence of dimerization. In particular, it was found that binding of ANSA to MB-1-Cys dimer was comparable to that observed for native, compact, natural proteins. We discuss the implications of these results for the design of transgenic protein production systems.