In this report, we describe a flexible, efficient and rapid protein purification strategy for the isolation and cleavage of glutathione-S-transferase (GST) fusion proteins. The purification and on-column cleavage strategy was developed to work for the purification of difficult proteins and for target proteins where efficient fusion-tag cleavage is essential for downstream processes, such as structural and functional studies. To test and demonstrate the flexibility of this method, seven diverse unrelated target proteins were assayed. A purification technique is described that can be applied to a wide range of both soluble and membrane inserted recombinant target proteins of differing function, structure and chemical nature. This strategy is performed in a single chromatographic step applying an on-column cleavage method, yielding "native" proteins in the 200 microg to 40 mg/l scale of 95-98% purity.