Pwur702

Company: Addgene

Catalog#: 53079

Preparation and Purification of β-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay

Pirjo Pärnänen, Timo Sorsa, Taina Tervahartiala and Pirjo Nikula-Ijäs

Published online: 2024-03-20

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Expression and Purification of the Thermus thermophilus Argonaute Protein

Author:

Daan C. Swarts, Matthijs M. Jore and John van der Oost,

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 expression of (toxic) TtAgo. Here, we describe the steps required for controlled expression and purification of this toxic protein. [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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