High-Level Expression of Milk-Derived Antihypertensive Peptide in Escherichia coli and Its Bioactivity

Abstract

An optimal antihypertensive peptide (AHP), KVLPVP, was linked to form a six-copy of tandem dotetracontapeptide with the specific cleavage site (Arg-X) of clostripain. The gene of the dotetracontapeptide was synthesized and expressed in Escherichia coli BL21. After a 5 h induction with 1.2 mM isopropyl-beta-D-thiogalactopyranoside the recombinant AHP fused with glutathione-S-transferase tag reached the maximal production, 24.6% of total intracellular protein. Following digestion with clostripain and carboxypeptidase B, the product was separated with ultrafiltration and reversed-phase HPLC, and 170 mg of pure recombinant AHP was obtained from 1 L of E. coli culture. The IC50 of the recombinant AHP was 4.6 microM. The systolic blood pressure of spontaneously hypertensive rats could be decreased dramatically by the recombinant AHP in a dose-dependent manner after delivering 0.3 mg of AHP/kg of body weight (BW) or 0.6 mg of AHP/kg of BW orally. The strong antihypertensive effect was reached 4-24 h after oral administration of 0.3 mg of AHP/kg of BW, and the peak point was at the fourth hour (-21.4 +/- 7.2 mm of Hg). This study overcame traditional enzymatic digestion problems in preparing AHP and established a novel approach for industrial production of AHP.