Mammalian alpha-defensins contribute to innate immunity by exerting antimicrobial activity against various pathogens. To perform structural and functional analysis of alpha-defensins, large amounts of alpha-defensins are essential. Although many expression systems for the production of recombinant alpha-defensins have been developed, attempts to obtain large amounts of alpha-defensins have been only moderately successful. Therefore, in this study, we applied a previously developed aggregation-prone protein coexpression method for the production of mouse alpha-defensin cryptdin-4 (Crp4) in order to enhance the formation of inclusion bodies in Escherichia coil expression system. By using this method, we succeeded in obtaining a large amount of Crp4 in the form of inclusion bodies. Moreover, we attempted to refold Crp4 directly during the inclusion-body solubilization step under oxidative conditions. Surprisingly, even without any purification, Crp4 was efficiently refolded during the solubilization step of inclusion bodies, and the yield was better than that of the conventional refolding method. NMR spectra of purified Crp4 suggested that it was folded into its correct tertiary structure. Therefore, the method described in this study not only enhances the expression of alpha-defensin as inclusion bodies, but also eliminates the cumbersome and time-consuming refolding step. (C) 2015 Elsevier Inc. All rights reserved.