Wang Junjie, Nishimura Mitsuhiro, Wakata Aika, Mori Yasuko
Division of Clinical Virology, Kobe University Graduate School of Medicine, Kobe, Japan.
Kobe J Med Sci. 2017 Apr 7;62(6):E142-E149.
Human herpesvirus 6A (HHV-6A) starts its replication cycle following the action of immediate early proteins that transactivate viral promoters. Immediate early protein 2 (IE2) of HHV-6A is a 1500 amino acid polypeptide with a C-terminal region that is conserved among beta-herpesvirus subfamily members. In this study, a structural domain in the homologous C-terminal region was subjected to secondary structure prediction, and residues 1324-1500 were subsequently designated as the C-terminal domain of IE2 (IE2-CTD). The gene fragment encoding IE2-CTD was inserted into an E. coli expression vector and expressed as a fusion protein with maltose binding protein (MBP) at the N-terminus. IE2-CTD has a theoretical isoelectric point (pI) of 9.29, and strong cation exchange column chromatography was effective for purification. Needle-shaped crystals of IE2-CTD were obtained using the sitting-drop vapour diffusion method, and larger selenomethionine-labelled crystals of space group P2₁ diffracted X-rays to 2.5 Å resolution using synchrotron radiation. Data were collected at the selenium absorption peak wavelength for experimental phasing by the single anomalous dispersion method. The resulting electron density map clearly shows the protein backbone, and full structural determination and refinement are in progress.
人类疱疹病毒6A（HHV - 6A）在即刻早期蛋白激活病毒启动子后开始其复制周期。HHV - 6A的即刻早期蛋白2（IE2）是一种由1500个氨基酸组成的多肽，其C末端区域在β疱疹病毒亚科成员中保守。在本研究中，对同源C末端区域的一个结构域进行了二级结构预测，随后将1324 - 1500位氨基酸残基指定为IE2的C末端结构域（IE2 - CTD）。将编码IE2 - CTD的基因片段插入大肠杆菌表达载体，并在N末端作为与麦芽糖结合蛋白（MBP）的融合蛋白进行表达。IE2 - CTD的理论等电点（pI）为9.29，强阳离子交换柱色谱法对其纯化有效。使用坐滴气相扩散法获得了IE2 - CTD的针状晶体，通过同步辐射，更大的P2₁空间群的硒代甲硫氨酸标记晶体将X射线衍射到2.5 Å分辨率。在硒吸收峰波长处收集数据，用于通过单异常色散法进行实验相位测定。所得电子密度图清晰显示了蛋白质主链，完整的结构测定和精修正在进行中。
