Brevinin-2GU, a member of a brevinin-2 family of antimicrobial peptides isolated from an extract of the skin of the Asian frog, Hylarana guntheri, displays significant insulin-releasing activity. In this study, in order to obtain relatively large quantity of functional brevinin-2GU protein, the coding sequence of brevinin-2GU gene was cloned into pE132a (+) vector and expression as a Trx fusion protein in Escherichia coli. The results indicated that the expression level of the fusion protein could reach up to 45% of the total cell proteins. the soluble fusion protein collected from the supernatant of the cell lysate was then separated by Ni2+-chelating chromatography and cleaved by Factor Xa protease to release mature brevinin-2GU. Our results showed that the final yield of the brevinin-2GU was 1.7 mg, the purified peptide displayed insulin-releasing activity similar to that of the purified brevinin that was reported earlier. This method allows production of sufficiently large amounts of brevinin-2GU peptide for functional and structural characterizations. (c) 2008, The Society for Biotechnology, Japan. All rights reserved.