Food-derived angiotensin converting enzyme inhibitory peptides (ACEIP) show a great promise for prevention and treatment of hypertension. Tuna muscle derived peptide, Tuna AI (PTHIKWGD), is a potent ACEIP with high activity and stability. To improve the expression level of Tuna AI in Escherichia coli, multimer genes of Tuna AI were assembled and expressed as fusion proteins by constructing the vectors of pET30-4nTunaAI (n=1, 2, 4 and 8). The results showed the multimer genes were successfully expressed in the constructed system, and their expression level improved significantly with increasing repeat degree. The E. coli harboring pET30-32TunaAI was selected for production of peptide based on its highest expression level and its fusion protein was suit for urea-washing purification. The peptide monomer was released from the fusion protein by formic acid cleavage, and successively purified by membrane filtration, cation exchange chromatography and size exclusion chromatography, with a final yield of 218.9 mg/L. The purified monomeric peptide was confirmed by mass spectrometry analysis, N-terminal sequencing, and activity measurement. This peptide resulted in a significant decrease of systolic blood pressure by 36.5 mmHg in rats at 4h. These results suggest our strategy is an economic solution for the mass production of antihypertensive peptides. (C) 2015 Elsevier Ltd. All rights reserved.