Recently, a durum wheat (Triticum durum Desf.) secretory phospholipase A(2) (TdsPLA(2)III) was identified in leaves as potentially involved in plant responses to conditions of limiting water supply. Therefore, to allow future functional studies on TdsPLA(2)III and shed further light on the involvement of sPLA(2) isoforms in specific plant functions, here we report a protocol for the overexpression of TdsPLA(2)III in Escherichia coli in the form of inclusion bodies, and for its purification and refolding. The use of the Gateway system (Invitrogen) allows the expression of a large quantity of the mature form (without the signal peptide) of TdsPLA(2)III with an N-terminal 6 x His-tag, for purification using Ni-affinity chromatography. The purified recombinant 6 x His-TdsPLA(2)III fusion protein is then refolded using a step-wise dialysis approach. About 40 mg purified and active protein was obtained from 1 L of cell culture. This recombinant 6 x His-TdsPLA(2)III protein shows PLA(2) activity, as it can hydrolyze linoleate from the sn-2 position of 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine. Moreover, it has some features that are typical of other known plant sPLA(2)s: Ca2+-dependence, inhibition by the disulfide bond reducing agent dithiothreitol, and resistance to high temperature. (C) 2014 Elsevier Inc. All rights reserved.