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.
Department of Chemistry, University of Prince Edward Island, Prince Edward Island, Canada.
J
J
Grundy
J J
JJ
Morrison
J D
JD
MacCallum
L
L
Wirtanen
M
M
Beauregard
D000595:Amino Acid Sequence; D003460:Crystallization; D004044:Dietary Proteins; D019281:Dimerization; D005453:Fluorescence; D006112:Gravitation; D006902:Hydroxymercuribenzoates / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D011506:Proteins; D013026:Space Flight; D013696:Temperature
D004044:Dietary Proteins; D006902:Hydroxymercuribenzoates; C107457:MB-1synthetic dietary protein; D011506:Proteins; C030601:4-hydroxymercuribenzoate