Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.
11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to approximately 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 A resolution and belonged to space group P6(3), with unit-cell parameters a=b=97.6, c=74.8 A, gamma=120.0 degrees. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress.
Tandang-Silvas
Mary Rose
MR
Laboratory of Food Quality Design and Development, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Carrazco-Peña
Laura
L
Barba de la Rosa
Ana Paulina
AP
Osuna-Castro
Juan Alberto
JA
Utsumi
Shigeru
S
Mikami
Bunzo
B
Maruyama
Nobuyuki
N
D027721:Amaranthus / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D015870:Gene Expression; D005916:Globulins / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D055314:Seed Storage Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D005916:Globulins; D010940:Plant Proteins; D055314:Seed Storage Proteins