An internal fragment (978 bp) corresponding to bonnet monkey (Macaca radiata) zone pellucida glycoprotein-3 (r-bmZP3), excluding the signal sequence and the transmembrane-like domain, was cloned in frame downstream of the T5 promoter under lac operator control in a pQE-30 vector. r-bmZP3 was expressed as a polyhistidine fusion protein in Escherichia coli strain BL21(pLysS), deficient in the ompT and ion proteases. The use of protease deficient host cells helped in reducing the level of premature translational termination products of r-bmZP3. It was expressed intracellularly as insoluble inclusion bodies. In shake flask culture, using Luria broth medium, the presence of 0.5% glucose enhanced the expression level of r-bmZP3. Based on information derived from shake flask cultures, a high cell density fermentation process was developed for large scale expression of the protein. After 10 h of fed-batch fermentation, a cell concentration of 25 g dry cell weight/l (A(600) of 65) and around 160 mg/l of the protein was expressed as inclusion bodies. Using a metal affinity column, 50 mg of the purified r-bmZP3 was obtained from 11 of fermentation broth as compared to 4 mg/l of shake flask culture. The purified protein was immunoreactive as tested by ELISA and Western blot, methods. (C) 2000 Elsevier Science Ltd. All rights reserved.