›› 2018, Vol. 61 ›› Issue (1): 59-67.doi: 10.16380/j.kcxb.2018.01.007

• 研究论文 • 上一篇    下一篇


苗娅, 张甜甜, 许柏英*, 陈斌*   

  1. (重庆师范大学昆虫与分子生物学研究所, 媒介昆虫重庆市重点实验室, 重庆 401331)
  • 出版日期:2018-01-20 发布日期:2018-01-20

Expression, purification and crystallization of the glutathione-S-transferase E6(AsGSTE6) from Anopheles sinensis (Diptera: Culicidae)

MIAO Ya, ZHANG Tian-Tian, XU Bo-Ying*, CHEN Bin*   

  1.  (Chongqing Key Laboratory of Vector Insects, Institute of Insect and Molecular Biology, Chongqing Normal University, Chongqing 401331, China)
  • Online:2018-01-20 Published:2018-01-20

摘要:  【目的】对中华按蚊Anopheles sinensis谷胱甘肽-S-转移酶E6(AsGSTE6)进行初步晶体学研究,为深入研究其空间结构奠定基础。【方法】利用生物信息学软件,首先对中华按蚊AsGSTE6进行生物信息学预测和分析;然后进行分子克隆,构建重组子pET28a-AsGSTe6;利用大肠杆菌Escherichia coli原核表达系统进行诱导表达;采用Ni-NTA金属螯合层析和葡聚糖凝胶层析的方法对重组蛋白进行纯化;通过葡聚糖凝胶层析和化学交联的结果分析蛋白的聚合状态;运用坐滴蒸气扩散法对重组蛋白进行结晶筛选。【结果】生物信息学分析结果表明,AsGSTE6分子量为25.28 kD,无信号肽和跨膜区,是亲水性蛋白;三级结构预测结果显示,AsGSTE6主要由一个小的βαβαββα折叠模式的N端结构域和一个大的全螺旋的C端结构域组成。多重序列比对结果表明,在不同昆虫中GSTE6具有高度保守性。分子克隆得到中华按蚊AsGSTE6的编码基因AsGSTe6序列,大小为672 bp。在大肠杆菌中AsGSTE6主要在上清中有大量表达,为可溶性表达;通过镍柱亲和层析和凝胶过滤层析纯化出了高纯度且稳定的AsGSTE6重组蛋白;凝胶过滤层析结合化学交联的结果证实体外纯化的融合蛋白AsGSTE6主要呈二聚体状态;通过晶体筛选获得了AsGSTE6的晶体。【结论】运用结晶学的方法获得了AsGSTE6的晶体,这为后续解析AsGSTE6的三维结构奠定了基础。

关键词: 中华按蚊, 谷胱甘肽S转移酶, AsGSTE6, 原核表达, 纯化, 结晶

Abstract: 【Aim】 This study aims to explore the structure of the glutathione-S-transferase E6 (AsGSTE6) from Anopheles sinensis based on its preliminary crystallography. 【Methods】 AsGSTE6 was predicted and analyzed by bioinformatics method. AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed and expressed in prokaryotic expression system. The recombinant protein was purified with nickel chelate affinity chromatography and gel filtration chromatography. The polymerization state of AsGSTE6 was detected by gel filtration chromatography and chemical crosslinking analysis. Crystal screening was performed by sitting drop vapor diffusion techniques. 【Results】 Bioinformatic analysis revealed that AsGSTE6 is a hydrophilic protein with the calculated molecular weight of 25.28 kD, without transmembrane regions and signal peptides in the amino acid sequence. The 3D structural prediction showed that AsGSTE6 contains a small N-terminal domain following a βαβαββα pattern and a large C-terminal all-α domain. The result of multiple sequence alignment demonstrated that GSTE6 proteins are highly conserved in insects. The 672 bp coding sequence of AsGSTe6 was cloned, and the recombinant plasmid pET28a-AsGSTe6 was constructed correctly. The recombinant protein AsGSTE6 was expressed in soluble form in Escherichia coli. And then the highly purified and stable protein was obtained with two step affinity chromatography. Gel filtration chromatography and chemical crosslinking analysis showed that AsGSTE6 mainly existed as dimer in vitro. Finally, the protein crystals of AsGSTE6 were obtained by crystal screening. 【Conclusion】 Crystals of AsGSTE6 have been obtained by methods of crystallography, which lays the foundation for 3D structure determination of AsGSTE6.

Key words: Anopheles sinensis, glutathione-S-transferases, AsGSTE6, prokaryotic expression, purification, crystallization