| Expression, Purification and Crystallization of the Herpesvirus Nuclear Egress Complex (NEC)
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Author:
Date:
2016-07-20
[Abstract] The protocol describes the production and crystallization of the soluble form of the nuclear egress complex (NEC) from Herpes simplex virus 1 and Pseudorabies virus. The NEC is a heterodimer that consists of conserved proteins UL31 and UL34. NEC oligomerization deforms the inner nuclear membrane around the capsid in infected cells, thereby mediating capsid budding into the perinuclear space during nuclear egress. We have successfully developed a protocol for large-scale preparation of highly pure NEC from two different viruses in a prokaryotic expression system, which enabled us to crystallize these viral protein complexes and determine their structures. This procedure may be adapted to purify and crystallize other soluble protein complexes.
[摘要] 该协议描述了来自单纯疱疹病毒1和伪狂犬病病毒的核出口复合物（NEC）的可溶形式的产生和结晶。 NEC是由保守蛋白UL31和UL34组成的异源二聚体。 NEC低聚使受感染细胞中的衣壳周围的内核膜变形，从而在核出口期间介导衣壳发芽进入核周空间。 我们已经成功地开发了一个协议，从大规模制备高纯度NEC从两种不同的病毒在原核表达系统，这使我们能够结晶这些病毒蛋白复合物和确定其结构。 该程序可适于纯化和结晶其它可溶性蛋白质复合物。
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| Preparation of Golgi Membranes from Rat Liver
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Author:
Date:
2014-11-20
[Abstract] This protocol details the isolation of enriched Golgi membranes from rat liver, using discontinuous density gradient centrifugation. This high-yield extraction method is useful for several applications, including immunoprecipitation of solubilised Golgi membrane proteins (preparation included) and electron microscopy. Protocol adapted from Leelavathi et al. (1970).
[摘要] 该协议详述了使用不连续密度梯度离心从大鼠肝脏分离富集的高尔基体膜。 这种高产率提取方法可用于几种应用，包括溶解的高尔基膜蛋白（包括制备）和电子显微镜的免疫沉淀。 从Leelavathi等人改编的方案（1970）。
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