High-level expression of milk-derived antihypertensive peptide in Escherichia coli and its bioactivity.
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.
Liu
Dong
D
Department of Applied Biotechnology, Shenzhen Polytechnic, Shenzhen, Guangdong 518055, People's Republic of China. liudongszpt@163.com
Sun
Haiyan
H
Zhang
Lijun
L
Li
Shimin
S
Qin
Zhenyu
Z
D000818:Animals; D000959:Antihypertensive Agents / Q000008:administration & dosage; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006973:Hypertension / Q000188:drug therapy; D007700:Kinetics; D008297:Male; D008892:Milk / Q000737:chemistry*; D010455:Peptides / Q000008:administration & dosage / Q000235:genetics*; D051381:Rats; D011918:Rats, Inbred SHR; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage
D000959:Antihypertensive Agents; D010455:Peptides; D011993:Recombinant Fusion Proteins