钙卫蛋白(CP,S100A8/S100A9杂寡聚物)是中性粒细胞表达的丰富的金属螯合宿主防御蛋白,其他白细胞,和上皮细胞。载脂蛋白是S100A8/S100A9异二聚体,其在S100A8/S100A9界面包含两个用于过渡金属结合的位点:His3Asp基序(位点1)和His6基序(位点2)。在这一章中,我们提供了过表达和纯化CP的人和鼠直系同源物的分步方案,其以高产率和纯度提供每个apo异二聚体.在这些程序中,S100A8和S100A9亚基在大肠杆菌BL21(DE3)中过表达,每个apo异二聚体在细胞裂解后获得,折叠,柱层析,并对Chelex树脂进行透析以减少金属污染。最近的研究表明,人类CP与Fe(II)配位,并且该蛋白质会影响溶液中Fe的氧化还原形态。描述了使用铁嗪的Fe氧化还原形态测定,该测定证明了CP的人和鼠直系同源物随时间将Fe的氧化还原形态从三价铁转变为亚铁氧化态的能力。
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.