Coated vesicle isolation by immunoadsorption on Staphylococcus aureus cells.

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Abstract
Porcine brain coated vesicles were isolated from crude fractions of tissue homogenates by affinity separation using anticlathrin-coated Staphylococcus aureus (Staph A) cells as a solid-phase immunoadsorbent. The specificity of the immunoadsorption was monitored by SDS [sodium dodecylsulfate] analysis and by competitive ELISA [enzyme linked immunosorbent assay] assays. SDS PAGE of the material immunoadsorbed from a fraction of porcine brain smooth microsomes showed a selective enrichment in a 180,000 MW protein. In an ELISA assay, this protein competed effectively-in binding anticlathrin-with clathrin extracted from a coated vesicle preparation. When the immunoadsorbed fraction was examined by EM, coated vesicles and vesicle-free cages were found forming a quasicontinuous monolayer on the surface of the Staph A cells. Other particles were not adsorbed, and the controls were free of either clathrin cages or coated vesicles. Upon extensive dialysis (against MES (2-[N-morpholino]ethane sulfonic acid) buffer, pH 6.5), similar cages appeared on the surface of anticlathrin-coated Staph A cells reacted with extracted clathrin. Apparently, anticlathrin-coated Staph A cells can be used for the isolation and purification of a homogeneous population of coated vesicles. The ability of extracted clathrin to bind and to polymerize onto the Staph A cells raises the possibility of using this technique to further explore the conditions required for cage and/or vesicle reconstitution.