Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.
Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.
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DOI：
10.1107/s1744309110021032
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发表时间：
2010-08
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影响因子：
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通讯作者：
M. R. Tandang-Silvas;Laura D. Carrazco‐Peña;A. P. Barba de la Rosa;J. A. Osuna-Castro;S. Utsumi;B. Mikami;N. Maruyama
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作者：
M. R. Tandang-Silvas;Laura D. Carrazco‐Peña;A. P. Barba de la Rosa;J. A. Osuna-Castro;S. Utsumi;B. Mikami;N. Maruyama
11S球蛋白是苋菜中主要的种子贮藏蛋白之一。利用含有pET21d和苋菜11S球蛋白cDNA的大肠杆菌Rosetta-gami （DE3）制备的重组蛋白约占细菌总蛋白的80%。最佳表达条件是在含0.5 M NaCl的LB培养基中，在302 K下培养20 h。用0-40%硫酸铵溶液沉淀，可以很容易地从大多数大肠杆菌蛋白中分离出重组蛋白。它在低温和低盐浓度下形成聚集体。这种行为可能意味着它比其他11S种子球蛋白具有更强的疏水性。晶体衍射分辨率为6 A，属于空间群P6(3)，晶胞参数A =b=97.6, c=74.8 A, γ =120.0度。假设Vsol为41%，估计三聚体的一个亚基存在于不对称单元中。为了获得完整的结构溶液，改进结晶和闪冷条件的实验正在进行中。
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.