Expression of antihypertensive peptide, His-His-Leu, as tandem repeats in Escherichia coli.
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.
Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea.
Do-Won
DW
Jeong
Dong Seok
DS
Shin
Chang-Won
CW
Ahn
In Sang
IS
Song
Hyong Joo
HJ
Lee
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D000959:Antihypertensive Agents / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000235:genetics*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D020080:Tandem Repeat Sequences
D000806:Angiotensin-Converting Enzyme Inhibitors; D000959:Antihypertensive Agents; D009842:Oligopeptides; D011994:Recombinant Proteins