The high level expression of recombinant proteins in Escherichia coli often leads to the formation of inclusion bodies that contain most of the expressed protein held together by non-covalent forces. The inclusion bodies are usually solubilized using strong denaturing agents like urea and guanidium hydrochloride. In this study recombinant Omp28 (rOmp28) protein of Brucella melitensis was expressed in two different vector systems and further efficient purification of the protein was done by modification in buffers to improve the yield and purity. Different concentrations of Triton X-100 and beta-mercaptoethanol were optimized for the solubilization of inclusion bodies. The lysis buffer with 8 M urea alone was not sufficient to solubilize the inclusion bodies. It was found that the use of 1% Triton X-100 and 20 mM beta-mercaptoethanol in lysis and wash buffers used at different purification steps under denaturing conditions increased the yield of purified rOmp28 protein. The final yield of purified protein obtained with modified purification protocol under denaturing conditions was 151 and 90 mg/l of the culture or 11.8 and 9.37 mg/g of wet weight of cells in pQE3OUA and pET28a(+) vector respectively. Thus modified purification protocol yielded more than threefold increase of protein in pQE3OUA as compared with purification by conventional methods. (C) 2012 Elsevier Inc. All rights reserved.