Recombinant protein purification remains to be a major challenge in biotechnology and medicine. In this paper we report a simple method for recombinant protein purification using self-assembling peptide tagged tobacco etch virus protease (TEVp). After construction of an N-terminal ELK16 peptide fusion expression vector, we expressed ELK16 TEVp fusion protein in E. coli. SDS-PAGE analysis showed that ELK16 TEVp was expressed as active protein aggregates which could be purified to 91% purity with 92% recovery by centrifugation in the presence 0.5% Triton X-100. By using His-tagged bovine interferon-gamma (His-BolFN-gamma) as the substrate, we demonstrated that EKL16-TEVp had a protease activity of 13 x 10(4) units/mg protein with almost 100% cleavage efficiency under the optimized conditions. More importantly, EKL16-TEVp could be removed from the cleavage reaction by single-step centrifugation. After removing the His-tag by nickel-conjugated agarose bead absorption, the recombinant BoIFN-gamma (rBoIFN-gamma) was purified to 98.3% purity with 63% recovery. The rBoIFN-gamma had an antiviral activity of 1.6 x 10(3) units/mg protein against vesicular stomatitis virus. These data suggest that ELK16 TEVp may become a universal tool for recombinant protein purification. (C) 2016 Elsevier Inc. All rights reserved.