• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文期刊>Current Science: A Fortnightly Journal of Research >CLONING AND CRYSTALLOGRAPHIC ANALYSIS OF HUMAN CARBONIC ANHYDRASE I
【24h】

CLONING AND CRYSTALLOGRAPHIC ANALYSIS OF HUMAN CARBONIC ANHYDRASE I

机译：人体碳酸酐酶I的克隆及晶体学分析

获取原文
获取原文并翻译 | 示例
           
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The cDNA encoding the human carbonic anhydrase I (hCAI) gene was prepared using a specific primer and total mRNA extracted from human erythroleukaemia (HEL) cell line. The cDNA was amplified by PCR and was cloned into the expression vector pET-3a. Nucleotide sequencing of the cloned gene showed the following amino acid changes: Val31Ile and Val218Ala, when compared to erythrocyte hCAI sequence. The gene was induced by IPTG, and the protein purified by affinity column chromatography was found to be as active as erythrocyte human carbonic anhydrase I (eHCAI). N-terminal amino acid sequencing of the purified protein revealed that two methionines at the N-terminus have not been cleaved off by processing in E. coli. The recombinant human carbonic anhydrase I (rHCAI) has been crystallized for the first time. These crystals belong to the trigonal space group P3(1)2 1 with unit cell dimensions of a = b = 120.2 Angstrom, and c = 88.8 Angstrom. The crystals contain two molecules in their asymmetric unit, Diffraction data to 2.2 Angstrom resolution has been collected using an imaging plate diffractometer. The structure has been determined by the molecular replacement method. The overall folding of the rHCAI is similar to that of native eHCAI, the RMSD for the superposition of 254 C-alpha atom pairs being 0.54 Angstrom. In contrast to orthorhombic crystals of eHCAI, crystals of rHCAI are extremely sensitive to X-rays. [References: 38]
机译：使用特异性引物和从人红白血病（HEL）细胞系提取的总mRNA制备编码人碳酸酐酶I（hCAI）基因的cDNA。通过PCR扩增cDNA，并将其克隆到表达载体pET-3a中。与红细胞hCAI序列相比，克隆基因的核苷酸测序显示以下氨基酸变化：Val31Ile和Val218Ala。该基因是由IPTG诱导的，经亲和柱层析纯化的蛋白质与红细胞人碳酸酐酶I（eHCAI）一样具有活性。纯化蛋白的N端氨基酸测序表明，在N端尚未通过在大肠杆菌中进行加工来切割掉两个蛋氨酸。重组人碳酸酐酶I（rHCAI）首次结晶。这些晶体属于三角形空间群P3（1）2 1，其晶胞尺寸为a = b = 120.2埃，c = 88.8埃。晶体在其不对称单元中包含两个分子，使用成像板衍射仪收集了至2.2埃分辨率的衍射数据。结构已通过分子置换法确定。 rHCAI的整体折叠与天然eHCAI的相似，其中254个C-α原子对的叠加的RMSD为0.54埃。与eHCAI的正交晶体相比，rHCAI的晶体对X射线极为敏感。 [参考：38]

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