HaloTag-directed magnetic immobilization of PAD4 for target-based screening of natural-product inhibitors.
Abstract
Target-directed screening of bioactive natural products is often limited by the instability, poor recoverability, and repeated preparation requirements of soluble recombinant enzymes. Here, we developed a reusable magnetic peptidyl arginine deiminase 4 (PAD4) screening platform based on site-directed HaloTag immobilization. Recombinant Halo-PAD4 was captured directly from crude lysate by chloroalkane-functionalized magnetic nanoparticles (MNPs-Cl) without an additional labor-intensive and time-consuming enzyme purification step, generating MNPs-Halo-PAD4 with retained catalytic activity and inhibitor responsiveness. Compared with soluble PAD4, the immobilized enzyme showed improved activity retention during storage and temperature exposure and could be magnetically recovered for repeated assays. The platform was applied to screen water, 75% ethanol, and ethyl acetate extracts of 15 traditional Chinese medicine (TCM) materials. Among the tested materials, Alpinia officinarum consistently exhibited the strongest PAD4-inhibitory activity in both soluble and immobilized PAD4 assays. Activity-guided high-performance liquid chromatography fractionation localized the inhibitory activity to fraction F3. Liquid chromatography-tandem mass spectrometry tentatively annotated 13 constituents, and authentic-standard testing prioritized galangin. Galangin inhibited PAD4 with an IC50 value of 46.6 μM and showed direct interaction with PAD4 by biolayer interferometry, with an apparent KD of 7.8 μM. Molecular docking further provided a structural hypothesis for the interaction. This workflow combines HaloTag-chloroalkane chemistry, magnetic enzyme immobilization, liquid chromatography and mass spectrometry for the screening of PAD4 inhibitors. This proposed one-step capture and immobilization of recombinant protein also supports ligand screening for other protein targets.
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