Part:BBa_K3142008
Artificially designed antihypertensive pentapeptide（KYLCY）
Antihypertensive active pentapeptide designed by Protein Structure Fingerprint Technology.The sequence has been optimized for lactic acid bacteria. It is a small molecule bioactive peptide that is difficult to directly biosynthesize. Therefore, the fusion expression strategy is currently used.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
In vitro activity detection of antihypertensive peptide(HPLC)
Principle
ACE can catalyze the decomposition of HHL to produce hippuric acid, while the blood pressure lowering peptide reduces the production of hippuric acid by inhibiting ACE activity, and the absorption of hippuric acid at 228 nm is determined by reverse phase HPLC to calculate the inhibition rate of the polypeptide of interest.
Method
Add 1.667×10-6moL/(s ∙ kg) ACE 25μL to the sample tube, normal tube and blank tube, respectively, 0.1 mol/L boric acid-borax buffer solution with a pH value of 8.3, 160, 170, 170 μL Add 10uL of blood pressure lowering peptide sample to the sample tube, and the other two tubes are not added and mixed. The blank tube was sterilized for 5 min at 100 ° C, and the other two tubes were reacted at 37 ° C for 5 min. Add 5 μL of HHL substrate to each tube, the total volume is 200 μL, and react at 37 ° C for 30 min. 100 ° C inactivated enzyme. Mobile phase composition:
- Phase A is 100% ACN plus 0.1% TFA
- Phase B is 0.1% TFA with 5% ACN.
Chromatographic conditions: flow rate 0.8ml / min, column temperature 30 ° C, elution gradient 30% ~ 60% (A phase) 30min, hip uric acid retention time is 4.1min.
The amino acid sequence of new design antihypertensive peptides
In vitro activity was evaluated by IC50, and the smaller the IC50, the higher the activity of blood pressure lowering. As shown in Fig. 1.The IC50 of KYLCY is 3.30×10-6 mg/mL.
Reference
- Yang, J. Protein Structure Fingerprint Technology. [J] Bioinform, Genomics, Proteomics. 2018, 3(2): 1-3.
- Chen heru, Xu zhicheng.Reversed-phase HPLC Determination of the in-vitro Inhibition Kinetics of Angiotensin-converting Enzyme Inhibitors. JOURNAL OF INSTRUMENTAL ANALYSIS. 1999，18(1)：69-71
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