Abstract: Wild-type and variant crystals of a recombinant enzyme β-d-glucan glucohydrolase from barley (Hordeum vulgare L.) were obtained by macroseeding and cross-seeding with microcrystals obtained from native plant protein. Crystals grew to dimensions of up to 500 x 250 x 375 µm at 277 K in the hanging-drops by vapour-diffusion. Further, the conditions are described that yielded the wild-type crystals with dimensions of 80 x 40 x 60 µm by self-nucleation vapour-diffusion in sitting-drops at 281 K. The wild-type and recombinant crystals prepared by seeding techniques achieved full size within 5-14 days, while the wild-type crystals grown by self-nucleation appeared after 30 days and reached their maximum size after another two months. Both the wild-type and recombinant variant crystals, the latter altered in the key catalytic and substrate-binding residues Glu220, Trp434 and Arg158/Glu161 belonged to the P43212 tetragonal space group, i.e., the space group of the native microcrystals was retained in the newly grown recombinant crystals. The crystals diffracted beyond 1.57-1.95 Å and the cell dimensions were between a = b = 99.2-100.8 Å and c = 183.2-183.6 Å. With one molecule in the asymmetric unit, the calculated Matthews coefficients were between 3.4-3.5 Å3.Da-1 and the solvent contents varied between 63.4% and 64.5%. The macroseeding and cross-seeding techniques are advantageous, where a limited amount of variant proteins precludes screening of crystallisation conditions, or where variant proteins could not be crystallized.
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Luang, S.; Cairns, J.R.K.; Streltsov, V.A.; Hrmova, M. Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme β-D-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis. Int. J. Mol. Sci. 2010, 11, 2759-2769.
Luang S, Cairns JRK, Streltsov VA, Hrmova M. Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme β-D-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis. International Journal of Molecular Sciences. 2010; 11(7):2759-2769.
Luang, Sukanya; Cairns, James R. Ketudat; Streltsov, Victor A.; Hrmova, Maria. 2010. "Crystallisation of Wild-Type and Variant Forms of a Recombinant Plant Enzyme β-D-Glucan Glucohydrolase from Barley (Hordeum vulgare L.) and Preliminary X-ray Analysis." Int. J. Mol. Sci. 11, no. 7: 2759-2769.