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

ArticleinJournal of Microbiology and Biotechnology 17(6):952-9 · July 2007with8 Reads
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.
    • "In this study, a mass of monomer peptide was obtained on the contribution of the efficient preparation of fusion protein and convenient release of monomer. According to previous studies, some protocols could be used for monomer peptide purification in this study, but most of them relied principally on reversed phase chromatography in HPLC [21,28,41]. It is clear that HPLC provides a good performance in separation, and this method is more suited for analysis not for preparation. "
    [Show abstract] [Hide abstract] ABSTRACT: 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 Escherichia 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 4 h. These results suggest our strategy is an economic solution for the mass production of antihypertensive peptides.
    Full-text · Article · Feb 2015
    • "This shortcoming has been conquered by expression of antihypertensive peptides in the forms of a fusion protein or a tandem gene. Antihypertensive peptides with sequences HHL, HVLPVP, FFVAPFPEVFGK, and GHIATFQER have been expressed successfully in Escherichia coli105106107108, although special proteases are needed to release the target active protein, thus increasing the cost of separation and purification after enzymatic hydrolysis. Recently, Rao et al. [109] expressed an antihypertensive peptide multimer, a common precursor of 11 kinds of antihypertensive peptides, and the release was confirmed by simulated gastrointestinal digestion. "
    [Show abstract] [Hide abstract] ABSTRACT: Bioactive food peptides are encrypted within the sequence of food proteins but can be released during food processing (by enzymatic hydrolysis or fermentation) or during gastrointestinal transit. Among bioactive food peptides, those with antihypertensive activity are receiving special attention due to the high prevalence of hypertension in the Western countries and its role in cardiovascular diseases. This paper reviews the current literature on antihypertensive food peptides, focusing on the main methodologies for their production, such as enzymatic hydrolysis, fermentation and the use of recombinant bacteria. This paper also describes the structure/activity relationship of angiotensin-converting enzyme (ACE)-inhibitory peptides, as well as their bioavailability, physiological effects demonstrated by both in vitro and in vivo assays, and the contribution of mechanisms of action other than ACE inhibition. Finally, current reported strategies for incorporation of antihypertensive peptides into foods and their effects on both availability and activity are revised in this manuscript.
    Full-text · Article · Dec 2010
  • [Show abstract] [Hide abstract] 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.
    Article · Sep 2009
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