J. Grundy et al., CRYSTALLIZATION AND STABILIZATION OF MB-1, A DE-NOVO DESIGNED PROTEINFOR OPTIMIZED FEEDING TECHNOLOGY, Journal of biotechnology, 63(1), 1998, pp. 9-15
Milk Bundle-1 is a de novo protein that was designed for application i
n agriculture. It has a high content of selected essential amino acids
, and is intended to adopt an alpha-helical bundle fold. Crystallizati
on experiments with MB-1 have been carried out on the ground and in re
duced gravity on board Columbia orbiter during mission STS-80. Rather
small crystals were obtained (< 0.05 mm) in both environments. Among o
ther factors, the lack of stability of purified MB-1 has been detrimen
tal to crystal growth. We report here on our progress with regard to o
ptimizing 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 o
ther mutant (MB-1-Cys) was engineered to carry a cysteine residue on a
solvent exposed face. The exposed cysteine binds readily to p-HMB, an
d allows for dimerization of MB-1-Cys. The dimer was found to be twice
as stable as MB-1 during proteolytic degradation studies. (C) 1998 El
sevier Science B.V. All rights reserved.