A new protein fusion system has been developed to generate free recombinant protein in a single affinity chromatographic step. The key component in the fusion is the catalytic core of sortase A from Staphylococcus aureus (SrtAc), which recognizes and cleaves the Thr Gly bond at an LPXTG sequence with moderate activity. The fusion here consists of an N-terminal HiS(6) tag, SrtAc, and an LPETG linker followed by protein of interest at the C-terminus. The fusion protein is expressed in Escherichia coli and purified by immobilized metal-ion affinity chromatography (IMAC). The immobilized fusion then undergoes on-column SrtAc-mediated cleavage at the LPETG site in the presence of Ca2+ and/or triglycine. The target protein with an extra N-terminal glycine is released from the fusion while the N-terminal portion remains bound to the column. Because the cleavage enzyme SrtAc is co-expressed as a fusion with the target protein, the purification system eliminates exogenous proteolysis. This purification approach is simple, robust, inexpensive, time saving, and allows purification of free recombinant protein via one-step chromatography. (C) 2004 Elsevier Inc. All rights reserved.