We have investigated the properties of variant pea seed protease inhibitors, homologous to the anti-carcinogenic Bowman-Birk inhibitor (BBI) from soybean but differing most significantly in amino acid sequences at the two independent sites of protease inhibition. The pea protease inhibitors were expressed, using Aspergillus niger, with yields of up to 23 mg secreted recombinant protein per litre of media. The recombinant proteins showed protease inhibitory activity and were deduced to be disulphide-bonded correctly; limited post-translational processing had occurred at the amino-terminal ends of all proteins. Differences in trypsin and chymotrypsin specific inhibitory activities, and in inhibition constants, were observed in studies of the two recombinant variants and BBI. (C) 2004 Elsevier Inc. All rights reserved.