Resuscitation promoting factor D (RpfD) is one of the five members of the Rpf-like family in Mycobacterium tuberculosis, which all have the resuscitation-promoting activity. Here, the complete rpfD gene was amplified and cloned into pDE22 expression vector. Then a recombinant form of soluble RpfD (sRpfD) was expressed in Mycobacterium vaccae. The soluble recombinant RpfD was purified by Ni-Sepharose affinity chromatography. The purified final product was >95% pure, and the molecular weight was 24.0 kDa, determined by 15% SDS-PAGE stained with Coomassie brilliant blue R-250. The yield of purification was about 1. 10 mg/L of the culture. The biochemical property of the sRpfD was analyzed by stimulating the resuscitation of avirulent M. tuberculosis H37Ra which was in "non-culturable" condition. The results indicate that the sRpfD from M. vaccae could more efficiently stimulate the resuscitation of M. tuberculosis H37Ra than the refolded recombinant RpfD (iRpfD) from Escherichia coli DH5 alpha, and that the rabbit anti-sRpfD serum could completely inhibit this resuscitation-promoting effect caused by these two kinds of recombinant RpfD. Our study indicates that this expression system may facilitate large-scale production and purification of sRpfD which have high biological activity for further functional, pharmacological and clinical investigations. (C) 2007 Elsevier Inc. All rights reserved.