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

• Published in: Journal of biotechnology Vol. 63; no. 1; pp. 9 - 15
• Main Authors: Grundy, J, Morrison, J.J, MacCallum, J.D, Wirtanen, L, Beauregard, M
• Format: Journal Article
• Language: English
• Published: Lausanne Elsevier B.V 30.07.1998; Amsterdam Elsevier; New York, NY
• Subjects: Affinity chromatography; Agri-food biotechnology; Amino Acid Sequence; Biological and medical sciences; Biotechnology; Crystal growth; Crystalline structure; CRYSTALLIZATION; Dietary Proteins; Dimerization; Dimers; ESSENTIAL AMINO ACIDS; FEED PROCESSING; Fluorescence; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Gravitation; Gravitational effects; Hydroxymercuribenzoates - metabolism; Methods. Procedures. Technologies; Models, Molecular; Molecular biophysics; Molecular cloning; Molecular Sequence Data; Mutation - genetics; PROTEIN CONCENTRATES; Protein Conformation; Protein design; Protein Engineering; Protein Folding; PROTEIN QUALITY; Protein stability; Protein structure; PROTEIN SYNTHESIS; Proteins; PROTEOLYSIS; Purification; Space Flight; Space life sciences; Structure in molecular biology; Temperature; Agri-food biotechnology; Asn (N), asparagine; EAA, essential amino acids; His (H), histidine; 3-D, three dimensional; EDTA, ethylenediaminetetraacetic acid; MWCO, molecular weight cut-off; p-HMB, p-hydroxymercuribenzoate; Protein stability; a.u., absorbance units; EGTA, ethylene glycol-bis( β-aminoethylether)- N, N, N′, N′-tetraacetic acid; SDS, sodium dodecyl sulfate; TE, tris EDTA; CAPE, Canadian Protein Crystallization Experiment; DTT, dithiothreitol; Proteolysis; PAGE, polyacrylamide gel electrophoresis; Protein design; Tyr, tyrosine; T, threonine; MW, molecular weight; Crystallization; L, leucine; K, lysine; PMSF, phenylmethylsulfonyl fluoride; Cys (C), cysteine; IPTG, isopropyl- β- d-thiogalactopyranoside; MBP, maltose binding protein; M, methionine; Protein structure; ANSA, 1-anilino-8-naphthalenesulfonic acid; MB-1, Milk Bundle-1; CD, circular dichroism; Temperature; Stability; Industrial application; Escherichia coli; Agricultural industry; Method; Space shuttle; Space research; De novo; Optimization; Protein; Operating conditions; Microorganism culture; Bacteria; Recombinant protein; Enterobacteriaceae
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/S0168-1656(98)00068-6

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 α-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.