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Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin

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Calcium-Binding Proteins of the EF-Hand Superfamily

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1929))

Abstract

Calprotectin (CP, S100A8/S100A9 heterooligomer) is an abundant metal-sequestering host-defense protein expressed by neutrophils, other white blood cells, and epithelial cells. The apoprotein is a S100A8/S100A9 heterodimer that contains two sites for transition metal binding at the S100A8/S100A9 interface: a His3Asp motif (site 1) and a His6 motif (site 2). In this chapter, we provide a step-by-step protocol for the overexpression and purification of the human and murine orthologues of CP that affords each apo heterodimer in high yield and purity. In these procedures, the S100A8 and S100A9 subunits are overexpressed in Escherichia coli BL21(DE3), and each apo heterodimer is obtained following cell lysis, folding, column chromatography, and dialysis against Chelex resin to reduce metal contamination. Recent studies demonstrated that human CP coordinates Fe(II) and that the protein affects the redox speciation of Fe in solution. An Fe redox speciation assay employing ferrozine is described that demonstrates the ability of both the human and murine orthologues of CP to shift the redox speciation of Fe from the ferric to the ferrous oxidation state over time.

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Acknowledgments

Our current studies of calprotectin are supported by the National Science Foundation (CHE-1352132) and the National Institutes of Health (R01GM118695 and R01GM126376).

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Rose C. Hadley & Elizabeth M. Nolan

Authors
  1. Rose C. Hadley
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  2. Elizabeth M. Nolan
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Corresponding author

Correspondence to Elizabeth M. Nolan .

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Editors and Affiliations

  1. Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Zürich, Switzerland

    Claus W. Heizmann

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Hadley, R.C., Nolan, E.M. (2019). Preparation and Iron Redox Speciation Study of the Fe(II)-Binding Antimicrobial Protein Calprotectin. In: Heizmann, C. (eds) Calcium-Binding Proteins of the EF-Hand Superfamily. Methods in Molecular Biology, vol 1929. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9030-6_25

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  • DOI: https://doi.org/10.1007/978-1-4939-9030-6_25

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9029-0

  • Online ISBN: 978-1-4939-9030-6

  • eBook Packages: Springer Protocols

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