LARGE-SCALE PURIFICATION OF SYNAPTOPHYSIN AND QUANTIFICATION WITH A NEWLY ESTABLISHED ENZYME-LINKED-IMMUNOSORBENT-ASSAY

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55-2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex, In order to achieve this, a conventional purificat ion procedure which consists of size exlusion chromatography, hydropho bic interaction chromatography and chromatofocusing has been developed . This procedure was compared with purification of SYP I by immunoaffi nity chromatography, The elution patterns of both procedures were moni tored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immuno blotting with SYP I-specific antibody. Contaminating proteins with rel ative molecular masses (M(r)) very similar to SYP I could be removed d uring the process of purification, demonstrating that the 38 kDa prote in found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I, A specific antiserum was raised in rabbits using a h ighly purified preparation of SYP I, This antiserum was used in combin ation with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and rel iable quantification of this hydrophobic membrane protein in all purif ication steps, starting with Triton X-100-lysed brain homogenates. Usi ng this ELISA, the concentration of SYP I in highly purified SSV was d etermined to be 5.8% of solubilized protein.