Preparation of isotopically labelled recombinant β-defensin for NMR studies

Emily S. Seo, Thomas Vargues, David J. Clarke, Dusan Uhrin, Dominic J. Campopiano

Research output: Contribution to journalArticlepeer-review

Abstract

beta-Defensins are a family of cationic peptides that contain six invariant cysteine residues that form characteristic disulfide bonds between Cys1-Cys5, Cys2-Cysand Cys3-Cys6. They have been shown to act as potent antimicrobial agents and chemokines. Human β-defensin 2 (HBD2) was first isolated from psoriatic skin lesions and the structure of this peptide has been solved by X-ray crystallography and NMR spectroscopy both of which are consistent with a fold that contains an N-terminal a-helix and three anti-parallel p-strands. Here, we report the expression and purification of the first isotopically labelled β-defensin (15N HBD2) with 100% incorporation of 15N using a recombinant Escherichia coli method. Multi, dimensional NMR spectroscopy experiments: 2D 1H-15N HSQC, 3D HSQC-TOCSY and 3D HSQC-NOESY for the assignment of resonances with no overlapping or ambiguous peaks. This isotopically labelled peptide is highly suitable for studying the interactions between HBD2 and a range of components from both the mammalian immune system and bacterial pathogens.

Original languageEnglish
Pages (from-to)179-184
Number of pages6
JournalProtein Expression and Purification
Volume65
Issue number2
DOIs
Publication statusPublished - Jun 2009

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