Open AccessExpression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.
Author(s) -
TandangSilvas Mary Rose,
CarrazcoPeña Laura,
Barba de la Rosa Ana Paulina,
OsunaCastro Juan Alberto,
Utsumi Shigeru,
Mikami Bunzo,
Maruyama Nobuyuki
Publication year - 2010
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s1744309110021032
Subject(s) - ammonium sulfate , amaranth , storage protein , crystallization , globulin , escherichia coli , recombinant dna , chemistry , trimer , protein subunit , germination , chromatography , food science , botany , biology , biochemistry , dimer , gene , organic chemistry , immunology
11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to ∼80% of the total bacterial protein using Escherichia coli Rosetta‐gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0–40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 Å resolution and belonged to space group P 6 3 , with unit‐cell parameters a = b = 97.6, c = 74.8 Å, γ = 120.0°. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a V sol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash‐cooling conditions are in progress.