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Microb Cell Fact. 2012 Apr 17;11:45.

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

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  • 1Agency for Science, Technology and Research, Nanos, Singapore.

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.

PMID:
22510322
[PubMed - indexed for MEDLINE]
PMCID:
PMC3447675
Free PMC Article
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