Preparation of isotopically labelled recombinant beta-defensin for NMR studies.
beta-Defensins are a family of cationic peptides that contain six invariant cysteine residues that form characteristic disulfide bonds between Cys(1)-Cys(5), Cys(2)-Cys(4) and Cys(3)-Cys(6). They have been shown to act as potent antimicrobial agents and chemokines. Human beta-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 alpha-helix and three antiparallel beta-strands. Here, we report the expression and purification of the first isotopically labelled beta-defensin ((15)N HBD2) with 100% incorporation of (15)N using a recombinant Escherichia coli method. Multidimensional NMR spectroscopy experiments: 2D (1)H-(15)N HSQC, 3D HSQC-TOCSY and 3D HSQC-NOESY allows 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.
Seo
Emily S
ES
School of Chemistry, The University of Edinburgh, King's Buildings, West Mains Road, Edinburgh, Lothian EH9 3JJ, United Kingdom.
Vargues
Thomas
T
Clarke
David J
DJ
Uhrín
Dusan
D
Campopiano
Dominic J
DJ
D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D012015:Reference Standards; D023083:beta-Defensins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
C106641:DEFB4A protein, human; D009587:Nitrogen Isotopes; D011993:Recombinant Fusion Proteins; D023083:beta-Defensins