...
  • 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Protein Expression and Purification >Expression, oxidative refolding, and characterization of six-histidine-tagged recombinant human LECT2, a 16-kDa chemotactic protein with three disulfide bonds
【24h】

Expression, oxidative refolding, and characterization of six-histidine-tagged recombinant human LECT2, a 16-kDa chemotactic protein with three disulfide bonds

机译：六组氨酸标签的重组人LECT2（具有三个二硫键的16-kDa趋化蛋白）的表达，氧化复性和表征

获取原文
获取原文并翻译 | 示例
   

获取外文期刊封面封底 >>

       
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Human LECT2 is a 16-kDa chemotactic protein that consists of 133 amino acids and three intramolecular disulfide bonds. Here, we present the oxidative refolding of (His)(6)-LECT2, an N-terminally (HiS)(6)-tagged recombinant protein of human LECT2. (His)(6)-LECT2 was overproduced in Escherichia coli in the form of insoluble aggregates, solubilized with 8 M urea in the presence of 10 mM DTT, and purified and refolded on Ni-NTA agarose by lowering the urea concentration before the elution. This process, however, gave a mixture of oligomers of (His)(6)-LECT2 as well as the monomer, whose composition was as low as 36%. The oligomers formed as a result of incorrect intermolecular disulfide bonds. After the refolding on Ni-NTA agarose (step 1), the disulfide bonds were shuffled using a glutathione redox buffer (step 2) and the remaining thiols were completely oxidized (step 3) to improve the yield of correctly folded, monomeric (His)(6)-LECT2. The monomer composition was significantly improved to 81% by the three-step refolding method and the monomer thus obtained was shown to have the same conformation as the authentic LECT2 produced in CHO cells by CD and NMR spectroscopies. The yield of (His)(6)-LECT2 was 1.0 mg/L E. coli culture and was 16 times as high as that in our previous report, in which (His)(6)-LECT2 was purified from the soluble fractions of E coli cell lysates. (C) 2002 Elsevier Science (USA). All rights reserved. [References: 15]
机译：人LECT2是一种16 kDa趋化蛋白，由133个氨基酸和三个分子内二硫键组成。在这里，我们介绍了人类His（6）-LECT2，N-末端（HiS）（6）-标签的人LECT2的重组蛋白的氧化重折叠。 （His）（6）-LECT2以不溶性聚集体的形式在大肠杆菌中过量生产，在10 mM DTT存在下用8 M尿素增溶，然后通过降低尿素浓度将其纯化并在Ni-NTA琼脂糖上折叠以降低洗脱前的浓度。但是，该方法得到了（His）（6）-LECT2的低聚物以及其组成低至36％的单体的混合物。由于不正确的分子间二硫键形成低聚物。在Ni-NTA琼脂糖上重折叠后（步骤1），使用谷胱甘肽氧化还原缓冲液（步骤2）将二硫键改组（其余步骤2），并将剩余的硫醇完全氧化（步骤3），以提高正确折叠的单体（His）的产率。 （6）-LECT 2。通过三步重折叠法，单体组成显着提高到81％，由此获得的单体具有与CD和NMR光谱法在CHO细胞中产生的真实LECT2相同的构象。 （His）（6）-LECT2的产量为1.0 mg / L大肠杆菌培养物，是我们以前报告中的（His）（6）-LECT2的纯化率的16倍，其中大肠杆菌细胞裂解物。 （C）2002 Elsevier Science（美国）。版权所有。 [参考：15]

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号