Purification of proctolin-binding proteins from the foregut of the insect Blaberus craniifer

A membrane protein that specifically binds the insect neuropeptide proctoli n was purified using standard chromatography from cockroach foregut membran es. Proctolin-binding sites were efficiently solubilized with either the no nionic detergent digitonin or the zwitterionic detergent Chaps, as indicate d by the specific binding of H-3-proctolin to solubilized samples. A solubi lized sample obtained from 1600 foregut membranes was subjected to a five-s tep chromatographic purification including chromatofocusing, anion-exchange and size-exclusion chromatographies. The final size-exclusion separation r esulted in the isolation of approximate to 100 pmol of purified proctolin-b inding proteins, eluting as a single peak at approximate to 74 kDa. Analysi s of the purified sample using SDS/PAGE and silver staining showed two band s at 80 kDa and 76 kDa. Densitometric analysis of the gel indicated that ea ch band contained approximate to 7-8 mu g of protein, suggesting that one b and corresponds to the proctolin-binding activity. Proctolin-binding protei ns were thus purified 1800-fold using standard chromatography.