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Int. J. Mol. Sci. 2010, 11(7), 2759-2769; doi:10.3390/ijms11072759
Article

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

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Received: 28 May 2010 / Revised: 16 July 2010 / Accepted: 16 July 2010 / Published: 19 July 2010
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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.
Keywords: macro- and cross-seeding; wild-type and mutant protein; X-ray diffraction macro- and cross-seeding; wild-type and mutant protein; X-ray diffraction
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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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.

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