Crystallization and stabilization of MB-1, a de novo designed protein for optimized feeding technology

Milk Bundle-1 is a de novo protein that was designed for application in agriculture. It has a high content of selected essential amino acids, and is intended to adopt an alpha-helical bundle fold. Crystallization experiments with MB-1 have been carried out on the ground and in reduced gravity on board Columbia orbiter during mission STS-80. Rather small crystals were obtained (< 0.05 mm) in both environments. Among other factors, the lack of stability of purified MB-1 has been detrimental to crystal growth. We report here on our progress with regard to optimizing crystal growth conditions, protein purification and protein stability. The first MB-1 mutant we present (MB-1-His) contains a poly-histidine tail, allowing the use of metal affinity chromatography for purification. MB-1-His has been found to keep its original mass for a month at room temperature, a spectacular improvement over MB-1. The other mutant (MB-1-Cys) was engineered to carry a cysteine residue on a solvent exposed face. The exposed cysteine binds readily to p-HMB, and allows for dimerization of MB-1-Cys. The dimer was found to be twice as stable as MB-1 during proteolytic degradation studies.