| 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 ...
[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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| Expression and Purification of the Thermus thermophilus Argonaute Protein
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Author:
Date:
2014-10-05
[Abstract] The Argonaute protein of Thermus thermophilus (TtAgo) has recently been studied in detail. For its in vitro characterization, TtAgo was purified after heterologous expression in Escherichia coli (E. coli). As TtAgo expression is toxic, a tightly controlled system was used for protein expression. The expression strain E. coli KRX carries a chromosomal T7 RNA polymerase gene under control of a rhamnose promoter. The ago gene is expressed via an IPTG-inducible T7 promoter. This allows for tightly (double) controlled ...
[摘要] 最近已经详细研究了嗜热栖热菌（Thermus thermophilus）（ Tt Ago）的Argonaute蛋白。 为了其体外表征，在大肠杆菌中大肠杆菌（ ）中异源表达后纯化Δtt。 作为Tt Ago表达是有毒的，严格控制的系统用于蛋白质表达。 表达菌株em。 大肠杆菌KRX携带在鼠李糖启动子控制下的染色体T7RNA聚合酶基因。 基因通过IPTG诱导型T7启动子表达。 这允许（有毒的）Tt Ago的紧密（双）受控表达。 在这里，我们描述了控制表达和纯化这种毒性蛋白所需的步骤。
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