Middleton Claire L, Parker Joanne L, Richard Derek J, White Malcolm F, Bond Charles S
Division of Biological Chemistry and Molecular Microbiology, School of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland.
Acta Crystallogr D Biol Crystallogr. 2003 Jan;59(Pt 1):171-3. doi: 10.1107/s0907444902019546. Epub 2002 Dec 19.
HolliDay junction endonuclease (Hje) from Sulfolobus solfataricus is a resolving enzyme involved in cleaving specific sites on either side of recombinant four-way HolliDay junctions. The HJE gene from S. solfataricus was cloned from genomic DNA into the pET19b Escherichia coli expression vector and recombinant protein was expressed to high levels. Hje was purified using heat treatment, cation exchange and gel filtration. Hanging-drop crystallization trials yielded primitive hexagonal crystals which diffract to 2.4 A on a laboratory source. Systematic absences (only 00l = 6n present) and poor scaling in P622 indicate that the space group is P6(1) or its enantiomer. Failed attempts at molecular replacement using models of a related archaeal resolving enzyme, Hjc, raise the possibility of a difference in quaternary structure between Hjc and Hje, which may be responsible for differences in their activities.
来自嗜热栖热菌的霍利迪连接内切酶（Hje）是一种参与切割重组四链霍利迪连接点两侧特定位点的解离酶。将嗜热栖热菌的HJE基因从基因组DNA克隆到pET19b大肠杆菌表达载体中，并高水平表达重组蛋白。使用热处理、阳离子交换和凝胶过滤对Hje进行纯化。悬滴结晶试验得到原始六方晶体，在实验室光源下其衍射分辨率为2.4 Å。系统消光（仅存在00l = 6n）以及在P622中的不佳缩放表明空间群为P6(1)或其对映体。使用相关古菌解离酶Hjc的模型进行分子置换的尝试失败，这增加了Hjc和Hje之间四级结构存在差异的可能性，这可能是它们活性差异的原因。
