Expression and Purification of the CMR (Type III-B) Complex in Sulfolobus solfataricus.
Expression and Purification of the CMR (Type III-B) Complex in Sulfolobus solfataricus.
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DOI：
10.1007/978-1-4939-2687-9_12
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发表时间：
2015
影响因子：
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通讯作者：
Jing Zhang;M. F. White
中科院分区：
文献类型：
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作者：
Jing Zhang;M. F. White
蛋白质纯化是一项重要的技术，它使我们能够表征单个蛋白质或多蛋白质复合物的结构和生化特性。然而，由于不稳定性和错误折叠等问题，在不存在其结合配偶体的情况下表达和纯化复合物的一个亚基通常被证明难以实现。CRISPR-Cas干扰复合物的组分就是这种情况，其在许多原核物种中以序列同源性依赖性方式降解入侵核酸。在这里，我们描述了一个串联标签的III-B型（CMR）复合体inSulfolobus solfataricus亚基的表达和随后的分离和纯化的整个复合体的亲和纯化的标签亚基。
Protein purification is an important technique that allows us to characterize the structural and biochemical properties of either an individual protein or a multi-protein complex. However, expression and purification of one subunit of a complex in the absence of its binding partners has often proven difficult to achieve due to the issues such as instability and mis-folding. This is the case for the components of the CRISPR-Cas interference complexes, which degrade invading nucleic acids in a sequence homology-dependent manner in many prokaryotic species. Here, we describe the expression of a tandem-tagged subunit of the Type III-B (CMR) complex inSulfolobus solfataricusand subsequent isolation and purification of the whole complex by affinity purification of the tagged subunit.