Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli

Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
Journal of Microbiology and Biotechnology (Impact Factor: 1.53). 07/2007; 17(6):952-9.
Source: PubMed


His-His-Leu (HHL), a tripeptide derived from a Korean soybean paste, is an angiotensin-I-converting enzyme (ACE) inhibitor. We report here a method of producing this tripeptide efficiently by expressing tandem multimers of the codons encoding the peptide in E. coli and purifying the HHL after hydrolysis of the peptide multiners. The HHL gene, tandemly multimerized to a 40-mer, was ligated with ubiquitin as a fusion gene (UH40). UH40 was inserted into vector pET29b; the UH40 fusion protein was then produced in E. coli BL21. The recombinant UH40 protein was purified by cation-exchange chromatography with a yield of 17.3 mg/l and analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and protein N-terminal sequencing. Leucine aminopeptidase was used to cleave a 405-Da HHL monomer from the UH40 fusion protein and the peptide was purified using reverse-phase high-performance liquid chromatography (HPLC) on a C18 HPLC column, with a final yield of 6.2 mg/l. The resulting peptide was confirmed to be HHL with the aid of MALDI-TOF mass spectrometry, glutamine-TOF mass spectrometry, N-terminal sequencing, and measurement of ACE inhibiting activity. These results suggest that our production method is useful for obtaining a large quantity of recombinant HHL for functional antihypertensive peptide studies.

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    ABSTRACT: To date, a number of antihypertensive peptides (AHPs) have been identified. Most of these are derived from proteins present in common edible consumables, including milk, egg, and plant foods. Consumption of these foods serves as means of AHP delivery and thus contributing favorable health benefits. It is hypothesized that food crops, either over-expressing AHP precursor proteins or producing particular peptides as heterologous components, may serve as viable vehicles for production and delivery of functional foods as alternative hypertension therapies. In recent years, genetic engineering efforts have been undertaken to add value to functional foods. Pioneering approaches have been pursued in several crop plants, such as rice and soybean. In this review, a summary of current tools used for discovery of new AHPs, as well as strategies and perspectives of capitalizing on these AHPs in genetic engineering efforts will be presented and discussed. The implications of these efforts on the development of functional foods for preventing and treating hypertension are also presented.
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    ABSTRACT: It is common for small tandem peptide multimer genes to be indirectly inserted into expression vectors and fused with a protein tag. In this study, a multimer of the tandem angiotensin I-converting enzyme inhibitory peptide (ACE-IP) gene was directly transferred to a commercially available vector and the designed gene was expressed as a repeated peptide in Escherichia coli BL21(DE3)pLysS. The process further developed in our study was the construction of six-repeated ACE-IP synthetic genes and their direct insertion. Protein expression in inclusion bodies was confirmed by SDS-PAGE and Western blot. Acid hydrolysis of inclusion bodies produced single-unit peptides through cleavage of the aspartyl-prolyl bonds. This cleaved recombinant peptide (rACE-IP) was purified using immuno-affinity chromatography followed by reversed phase-HPLC. 105-115 mg of the lyophilized recombinant peptide was obtained from 1 L E. coli culture. In vitro biological activity of rACE-IP was indistinguishable from that of the natural peptide produced by hydrolysis in artificial gastric juice or by acidic hydrolysis. The rACE-IP prepared by recombinant DNA technology and solid-phase synthesis methods showed a similar IC(50). This strategy could be used for the expression of important peptides, which have N-terminal proline (P) and C-terminal aspartic acid residues (D) for commercial applications, e.g. functional foods and drinks.
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