Isolation of functional endogenous clathrin-coated vesicles and in vitro reconstitution of pure clathrin cages

Clathrin-coated vesicles (CCVs) mediate key endocytic trafficking events, and dysfunction of CCVs is linked to multiple human diseases. Conventional centrifugation-based purification protocols can enrich endogenous CCVs but fail to eliminate intraluminal smooth vesicles (SVs) and co-isolated exosomes; these lipid contaminants reduce sample homogeneity and hinder high-resolution structural and proteomic analysis. Here, we present an integrated workflow to isolate biologically active endogenous CCVs from porcine brain and reconstitute homogeneous, SV-free clathrin cages. Crude CCVs are first enriched by differential and sucrose density gradient centrifugation to deplete the majority of exosomes. Clathrin cages are then depolymerized under alkaline conditions, allowing separation of soluble clathrin and adaptor proteins from SVs and residual lipid contaminants via ultracentrifugation. Purified coat proteins are reassembled in vitro into uniform clathrin cages. Functional assays confirm that purified CCVs retain Auxilin-Hsc70-dependent uncoating activity, and the enclosed SVs carry active vATPase proton pumps. This approach produces well-defined samples suitable for cryo-electron microscopy and mass spectrometry-based investigation of clathrin assembly and disassembly.