Screening Of Antihypertensive Peptide And Design, Cloning, Expression And Activity Identification Of Its Precursor Polypeptide

Hypertension is currently one of the most common cardiovascular diseases and growing gradually serious around the world. Chemically synthesized drugs are now broadly used to treat and prevent hypertension. Although having obvious antihypertensive effect, these drugs are reported to have many side effects and thus harm to human health. Food protein-derived antihypertensive peptides （AHPs） have received considerable attention, to their warm antihypertensive effects, safety, reliability, absorbability and no-effect on normal blood pressure, thereby becoming a hot topic for controlling and treating hypertension. For the last four decades, though near one thousand AHPs were produced and identified from enzymatic hydrolysates of food-derived proteins, low content in their parent proteins, high cost and low yield after enzymatic hydrolysis greatly hanger their commercialization. This study focuses on gene expression of AHPs to prepare AHPs on a large scale via molecular biology and microbial fermentation. This work provides a new strategy to produce a large quantity of AHPs, which makes up for the shortage of enzymatic hydrolysis method and paves the way for industrialization of AHPs. Considering gene expression efficiency in E. coli and recovery rate of active peptides and their release efficiencies from their designed precursor polypeptides, the corresponding solutions were put forward and conducted in this study, and the major results are as follows:1. 11 kinds of AHP monomers, including VWIS, VW, RIY, IY, LW, IKW, LKP, LKPNM, RPLKPW, NMAINPSK and IPP were tandemly linked up into antihypertensive peptide multimer-1 （AHPM-1） according to the restriction sites of gastrointestinal proteases. Based on the target favored codons of E. coli, the DNA fragment Ahpm-1 encoding the AHPM-1 was chemically synthesized and cloned into expression vector pGEX-3X. The recombinant strain E. coli BL21（DE3）/pGEX-3X-Ahpm-1 was constructed, and the aimed polypeptide AHPM-1 was successfully expressed as GST-AHPM-1 fusion protein. The culture condition for production of GST fusion protein, expressed in the form of inclusion body, was further optimized and the optimum condition was following: when OD₆₀₀ value before induction was 0.6～0.8, the expression of the target protein was induced by the addition of IPTG at a final concentration of 0.4 mmol/L. After incubation for 5 h at 30oC under this condition, GST fusion protein accounted for 35% of total intracellular protein. The inclusion body was washed, dissolved, and purified by IEC chromatography, followed by refolding together with SEC and gradual dialysis. The resulting yield of the soluble GST fusion protein with a purity of 95% reached 399 mg/L culture, and the recovery rate was 58.3%. Under simulated physiological condition, in vitro digestion of GST fusion protein was conducted using gastrointestinal enzymes and ACE, and the obtained final hydrolysate possessed potent ACE inhibitory activity with an IC₅₀ value of 35.2μg/mL. The results suggested that the high active fragments were successfully released from the AHPM-1 using gastrointestinal enzymes and ACE. From this respect, both the rationality of design of AHPM and the feasibility of production of AHPs by means of genetic engineering are confirmed in this study.2. To solve the problem that AHPM is easy to form inclusion body and further improve the potential ACE inhibitory activity of the designed multimer, 15 AHP monomers, including VK, AVPYPQR, IKP, YQEPVL, IKW, FALPQY, IVY, IFVPAF, KVLPVP, DGL, GVYPHK, IMY, GPL YPK and IPP, were rechosen and tandemly multimerized to AHPM-2 using a series of tandem strategies of active peptide monomers. The corresponding polypeptide gene Ahpm-2 was optimized using gene optimization software“Synthetic Gene Designer”. Afterwards, the optimized gene was cloned into downstream of the Trx （high hydrophilic tag, pI=5.27） gene in the expression vector pET32a, and then the AHPM-2 was successfully expressed in the form of soluble Trx-AHPM-2 fusion protein in E. coli Origami （DE3）. The optimum fermentation condition is following: when OD₆₀₀ value before induction was 0.6～0.8, the expression of the target protein was induced by the addition of IPTG at a final concentration of 0.1 mmol/L. After incubation for 5 h at 37 oC under this condition, the expression level of Trx fusion protein accounted for above 50% of total intracellular protein. The fusion protein was expressed above 90% in a soluble form. After Ni²⁺ affinity chromatographic （AC） purification followed by a size exclusion chromatography （IEC）, about 200 mg of Trx-AHPM-2 was obtained from 1 L culture of induced cells with 95% purity in the pooled fractions. The protein was subjected to a simulated gastrointestinal digestion, and the resulting hydrolysate exhibited potent ACE inhibitory activity with an IC₅₀ value of 4.5μg/mL. The results from UPLC-MALDI-TOF-TOF analysis suggest that the active hexapeptides with Pro in C-antepenultimate position and aromatic amino acids （Phe, Tyr or Trp） or Leu in C-terminus or N-terminus, are easy to be released from the precursor by action of pepsin, such as FALPQY and IFVPAF in our research. However, the active fragments with a C-terminal Pro, such as IKP, is not easy to be released from AHPM-2. In addition, when the distance of two Pro respectively lying in two adjacent sequences is too close, these two sequences are hard to be released from their precursor.3. Based on the selectivity of ACE on C-terminal dipeptides in its substrate, six pentapeptides, with the same tripeptide IKP （as a model） at N-terminus including IKPVQ, IKPVA, IKPVK, IKPVR, IKPFR, and IKPHL, were designed and chemically synthesized to solve the problem that the peptides with a Pro at C-terminus were hard to release from their precursors. The IC₅₀ values of these pentapeptides were all below 1.5μmol/L. Through UPLC-MADIL-TOF-TOF analysis, the ACE incubation experiment showed that IKP could be released from these pentapeptides by ACE, and the release rates were 23%～84.6%. The results demonstrated that the Pro-Val and Pro-Phe peptide bonds were possibly easily cleaved by ACE as compared with Pro-His bond. Meanwhile, the C-terminal basic amino acid residue can significantly improve the cleavage efficiency of ACE. Through UPLC-MADIL -TOF-TOF analysis, the in vitro digestion experiment revealed that all of the pentapeptides were resistant to peptic hydrolysis but except IKPVA with a retention rate of 80.5% not to pancreatic hydrolysis. Notably, the peptide IKPFR was hydrolyzed into IKPF (IC₅₀=4.6μmol/L) and IKP (IC₅₀=0.68μmol/L) by pancreatic digestion, and the corresponding hydrolysate still maintained a potent ACE inhibitory activity (IC₅₀=2.01μmol/L). IKP accounted for 37.9% of total peptides in the hydrolysate, and 62.1% for IKPF. Kinetics studies suggested that both IKPVA and IKPFR were all competitive inhibitors, which can competitively interact with the active sites of ACE instead of natural substrates. The results imply that IKPVA and IKPFR can be used to construct AHPM and then hydrolyzed to release IKP by ACE or gastrointestinal enzymes. Furthermore, the dipeptides VA and FR described here may be widely used as linkers to help the release of the active peptides with Pro at C-terminus from their polypeptide precursors by ACE or gastrointestinal enzymes in human body.4. The objective of the study was to screen the active peptide monomers, which possessed advantages of resisting physiological digestion and easily releasing from their precursor polypeptides by gastrointestinal enzymes or ACE. In this respect, the egg white lysozyme, whose sequence contains high proportion of hydrophobic （43.4%） and basic （13.7%） amino acid residues, was chose and subjected to a simulated physiological digestion. The resulting hydrolysate exhibited a potent ACE inhibitory activity with an IC₅₀ value of 15.6μg/mL Three novel ACE inhibitory peptides, MKR, RGY and VAW respectively with IC₅₀ values of 25.7, 61.9 and 2.86μmol/L, were purified from the gastrointestinal digests of egg white lysozyme by ultrafiltration, preparative RP-HPLC, analytical RP-HPLC and identified by MALDI-TOF-TOF. All of these active peptides identified in this study were first reported. Kinetics studies suggested that the purified peptides were all competitive inhibitors. ACE preincubation experiments implied that the peptide RGY was a true substrate and that the other two peptides were true ACE inhibitor. The results demonstrate that the peptides identified in this study are expected to be referred as active peptide monomers to design AHPM. Also, this work announces the importance of combination choose of protein staff and enzyme to produce the target peptides.