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使用酮类固醇异构酶标签表达和纯化 p75 神经营养因子受体跨膜结构域。

Expression and purification of the p75 neurotrophin receptor transmembrane domain using a ketosteroid isomerase tag.

机构信息

Agency for Science, Technology and Research, Nanos, Singapore.

出版信息

Microb Cell Fact. 2012 Apr 17;11:45. doi: 10.1186/1475-2859-11-45.

DOI:10.1186/1475-2859-11-45
PMID:22510322
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3447675/
Abstract

BACKGROUND

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

RESULT

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

CONCLUSIONS

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.

摘要

背景

具有单个跨膜 (TM) 结构域的受体对于跨细胞膜的信号转导至关重要。NMR 光谱学是研究单个 TM 结构域结构的有力工具。由于其分子量小，在大肠杆菌 (E.coli) 中表达和纯化 TM 结构域具有挑战性。尽管酮固醇异构酶 (KSI) 是表达和纯化短肽常用的亲和标签，但 KSI 标签需要用有毒试剂溴化氰 (CNBr) 去除。

结果

本文描述了使用带有凝血酶切割位点的 KSI 标签纯化 p75 神经营养因子受体的 TM 结构域。重组融合蛋白被重折叠成胶束并用凝血酶切割。研究表明，纯化的蛋白质可用于使用 NMR 光谱学进行结构研究。

结论

这些结果为获得用于结构研究的单个 TM 结构域提供了另一种策略，无需使用有毒化学消化或酸去除融合标签。p75 神经营养因子受体的纯化 TM 结构域将有助于结构研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/bae30ee333cb/1475-2859-11-45-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/90b11a61e875/1475-2859-11-45-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/ecc2f60549e7/1475-2859-11-45-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/d864f1b82143/1475-2859-11-45-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/da43381f4ae9/1475-2859-11-45-4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/261cc0a5df14/1475-2859-11-45-5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/bae30ee333cb/1475-2859-11-45-6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/90b11a61e875/1475-2859-11-45-1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/ecc2f60549e7/1475-2859-11-45-2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/d864f1b82143/1475-2859-11-45-3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/da43381f4ae9/1475-2859-11-45-4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/261cc0a5df14/1475-2859-11-45-5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a360/3447675/bae30ee333cb/1475-2859-11-45-6.jpg

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