A new systematic strategy for the isolation of proteins, illustrated by the purification of a mammalian exo-β-N-acetyl-d-glucosaminidase

Abstract

The ‘B’ form of the exo-β-N-acetyl-d-glucosaminidase from pig epididymis was purified by a new systematic strategy to yield a preparation with a specific activity at least equal to that obtainable by an existing empirically derived procedure. The new strategy is essentially a sequence of three carefully chosen steps consisting of an initial fractionation of the constituent proteins according to molecular size, followed by an ion-exchange step designed to select out proteins with closely similar electric-charge properties to those of the protein of interest, and a final high-resolution step dependent on the isoelectric points of the residual proteins. Gel isoelectric focusing itself, or as an element in the technique described by Leaback & Robinson (1974) for the separate display of the molecular size and electric-charge characteristics of proteins, played an important part in the choice of the experimental conditions used in the new strategy, and also in monitoring the progress of the purification.