Preparation of isotopically labelled recombinant β-defensin for NMR studies

• Published in: Protein expression and purification 2009-06, Vol.65 (2), p.179-184
• Main Authors: Seo, Emily S., Vargues, Thomas, Clarke, David J., Uhrín, Dušan, Campopiano, Dominic J.
• Format: Article
• Language: English
• Subjects: 15N; Antimicrobial peptide; beta-Defensins - biosynthesis; beta-Defensins - chemistry; beta-Defensins - isolation & purification; Crystallography, X-Ray; Defensin; Escherichia coli - genetics; Escherichia coli - metabolism; Humans; Isotope Labeling; Isotopic label; Magnetic Resonance Spectroscopy; Mass Spectrometry; Nitrogen Isotopes; NMR; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - isolation & purification; Reference Standards
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2008.11.007

β-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 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 α-helix and three antiparallel β-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. Multidimensional NMR spectroscopy experiments: 2D 1H– 15N 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.