降血压活性肽的筛选及其前体多肽的设计、克隆表达与活性鉴定
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摘要
高血压是目前最为常见的心血管疾病之一,在世界范围内呈日趋严重的态势。尽管化学合成类的西药具有很明显的降压效果,但会引起一系列的副反应,危害健康。源于食品蛋白质的降血压肽因其降压温和、安全可靠、易于吸收、且对正常血压无影响等优点而备受关注,成为控制和治疗高血压的研究热点。近四十年来,国内外研究学者已通过酶法水解食源性蛋白制备并鉴定出近千种降血压肽,但由于这些活性肽段在其母体蛋白序列中所占的比例小、酶解制备后期的分离成本高、产率低等极大地限制了降血压肽的工业化制备。本论文主要围绕降血压肽的基因表达研究来展开,宗旨是通过分子生物学手段和微生物发酵技术批量制备降血压肽,以弥补酶法制备降血压肽的不足,奠定降血压肽的产业化基础。本论文针对降血压肽的基因表达效率、表达产物的回收、表达产物中活性肽单体的释放等问题,提出了相应的解决方案和思路。主要研究结果如下:
1.从自行构建的活性肽数据库中选择了11种活性肽单体(VWIS﹑VW﹑RIY﹑IY﹑LW﹑IKW﹑LKP﹑LKPNM﹑RPLKPW﹑NMAINPSK与IPP),根据胃肠消化酶的水解特性将这些活性肽单体串联设计成降血压肽多聚体AHPM-1。采用大肠杆菌最优密码子,化学合成多聚体相应的基因Ahpm-1并克隆入表达载体pGEX-3X,构建重组菌株E. coli BL21(DE3) / pGEX-3X-Ahpm-1,目标多肽AHPM-1以GST融合蛋白的形式获得成功表达;进一步优化了GST融合蛋白的包涵体表达条件:诱导前OD_(600)值为0.6～0.8,0.4 mmol/L IPTG,30℃,诱导5 h。该条件下,目标蛋白达到了胞内总蛋白的35%。包涵体经洗涤、溶解、IEC纯化、SEC部分复性及梯度透析完全复性后,从1 L发酵液中获得了399 mg纯度95%以上的可溶性GST融合蛋白,回收率为58.6%。体外消化模拟实验显示GST融合蛋白经胃肠酶和ACE消化后的水解液具有很强的ACE抑制活性(IC_(50)=35.2μg/mL),表明AHPM-1在胃肠酶及ACE的作用下成功释放出了活性片段,以此证实降血压肽多聚体设计的合理性及基因工程法制备降血压肽的可行性。
2.为了解决降血压肽多聚体易形成包涵体的问题及进一步提高多聚体的潜在ACE抑制活性,重新选择了15种活性肽单体(包括VK﹑AVPYPQR﹑IKP﹑YQEPVL﹑IKW﹑FALPQY﹑IVY﹑IFVPAF﹑KVLPVP﹑DGL﹑GVYPHK﹑IMY﹑GPL﹑YPK和IPP),采用一系列的活性肽单体串联策略设计了多聚体AHPM-2,并使用“Synthetic Gene Designer”软件对相应的多肽基因Ahpm-2进行了优化。随后,将优化基因克隆入表达载体pET32a中高亲水酸性标签Trx(pI=5.27)基因的下游,目标多肽AHPM-2以Trx融合蛋白的形式在E.coli Origami (DE3)中成功获得可溶表达。适宜诱导条件为:诱导前OD_(600)值0.6～0.8,0.1 mmol/L IPTG,37℃,诱导5 h。该条件下,表达的Trx融合蛋白占到胞内总蛋白的50%以上,其中90%以上以可溶的形式表达。经镍柱和凝胶柱纯化后,目标蛋白的纯度达到95%以上,并可从1 L发酵液中获得约200 mg高纯Trx-AHPM-2。Trx融合蛋白经胃肠模拟消化后的水解液具有极强的ACE抑制活性(IC_(50)=4.5μg/mL)。UPLC-MALDI-TOF-TOF分析表明AHPM-2中带有Pro残基(处在倒数第三位)且C-末端或N-末端为芳香族氨基酸(Trp、Tyr和Phe)或Leu残基的活性六肽(如IFVPAF和FALPQY)易于胃蛋白酶高效率释放,但是C-末端为脯氨酸(Pro)残基的活性肽段(如IKP)难以从多聚体中释放,并且两条活性肽序列中各自带有的Pro残基之间的距离靠得太近也不利于活性肽序列的完整释放。
3.针对降血压肽多聚体中C-末端为Pro残基的高活性肽段难以释放的问题,研究根据ACE对其底物C-末端二肽的选择性,以IKP为模式三肽,设计并合成了六条ACE抑制五肽IKPVQ、IKPVA、IKPVK、IKPVR、IKPFR和IKPHL,其IC_(50)值均在1.5μmol/L以下。UPLC-MADIL-TOF-TOF质谱分析证实这些五肽均能在ACE的作用下释放IKP,释放率从23%到84.6%。另外,五肽中的Pro-Val和Pro-Phe肽键相对于Pro-His肽键更易于ACE的切割,且底物肽C-末端的碱性氨基酸残基能显著提高切割效率。通过胃肠消化模拟实验后,质谱分析表明这六条五肽均能抗胃蛋白酶的消化,但除了IKPVA(IC_(50)=0.94μmol/L)对胰酶具有80%的抗性外,其它五肽均被胰酶降解,其ACE抑制活性受到不同程度的影响。其中,IKPFR (IC_(50)=0.33μmol/L)在胰酶的作用下产生62.1%的IKPF(IC_(50)=4.6μmol/L)与37.9%的IKP(IC_(50)=0.68μmol/L),仍然具有很强的ACE抑制活性(IC_(50)=2.01μmol/L)。抑制动力学研究显示IKPVA和IKPFR均为竞争性抑制剂,能与ACE的天然底物竞争结合ACE的活性位点。以上结果表明IKPVA和IKPFR可用于构建降血压肽多聚体,并能在ACE或胃肠酶的作用下释放出高降压活性肽IKP。此外,二肽VA和FR有望作为连接片段用以辅助其它C-末端为Pro残基的活性肽段从其前体多肽中高效率释放。
4.为了筛选出既本身抗胃肠酶消化,又能被胃肠酶或ACE从其前体多肽中高效率释放的活性肽单体,研究选择了蛋白序列中具有高比例疏水性氨基酸(43.4%)和碱性氨基酸(13.7%)残基的蛋清溶菌酶进行胃肠消化模拟实验,发现其胃肠酶水解液发挥了很强的ACE抑制活性,IC_(50)值达到了15.6μg/mL。通过超滤、制备型RP-HPLC、分析型RP-HPLC及MALDI-TOF-TOF从溶菌酶的胃肠酶水解液中分离鉴定出三种ACE抑制肽MKR、RGY和VAW,其IC_(50)值分别为25.7﹑61.9和2.86μmol/L,均为首次报道。ACE温育实验结果表明VAW和MKR为真实型的ACE抑制肽,而RGY为底物型ACE抑制肽。抑制动力学研究显示VAW、MKR和RGY均为竞争性抑制剂。以上结果表明该研究鉴定的ACE抑制肽有望被作为设计降血压肽多聚体的活性肽单体,同时也揭示了蛋白原料和酶的组合选择对于筛选适宜目标肽段的重要性。
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 OD_(600) 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 IC_(50) 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 OD_(600) 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 Ni~(2+) 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 IC_(50) 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 IC_(50) 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 (IC_(50)=4.6μmol/L) and IKP (IC_(50)=0.68μmol/L) by pancreatic digestion, and the corresponding hydrolysate still maintained a potent ACE inhibitory activity (IC_(50)=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 IC_(50) value of 15.6μg/mL Three novel ACE inhibitory peptides, MKR, RGY and VAW respectively with IC_(50) 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.
1.从自行构建的活性肽数据库中选择了11种活性肽单体(VWIS﹑VW﹑RIY﹑IY﹑LW﹑IKW﹑LKP﹑LKPNM﹑RPLKPW﹑NMAINPSK与IPP),根据胃肠消化酶的水解特性将这些活性肽单体串联设计成降血压肽多聚体AHPM-1。采用大肠杆菌最优密码子,化学合成多聚体相应的基因Ahpm-1并克隆入表达载体pGEX-3X,构建重组菌株E. coli BL21(DE3) / pGEX-3X-Ahpm-1,目标多肽AHPM-1以GST融合蛋白的形式获得成功表达;进一步优化了GST融合蛋白的包涵体表达条件:诱导前OD_(600)值为0.6～0.8,0.4 mmol/L IPTG,30℃,诱导5 h。该条件下,目标蛋白达到了胞内总蛋白的35%。包涵体经洗涤、溶解、IEC纯化、SEC部分复性及梯度透析完全复性后,从1 L发酵液中获得了399 mg纯度95%以上的可溶性GST融合蛋白,回收率为58.6%。体外消化模拟实验显示GST融合蛋白经胃肠酶和ACE消化后的水解液具有很强的ACE抑制活性(IC_(50)=35.2μg/mL),表明AHPM-1在胃肠酶及ACE的作用下成功释放出了活性片段,以此证实降血压肽多聚体设计的合理性及基因工程法制备降血压肽的可行性。
2.为了解决降血压肽多聚体易形成包涵体的问题及进一步提高多聚体的潜在ACE抑制活性,重新选择了15种活性肽单体(包括VK﹑AVPYPQR﹑IKP﹑YQEPVL﹑IKW﹑FALPQY﹑IVY﹑IFVPAF﹑KVLPVP﹑DGL﹑GVYPHK﹑IMY﹑GPL﹑YPK和IPP),采用一系列的活性肽单体串联策略设计了多聚体AHPM-2,并使用“Synthetic Gene Designer”软件对相应的多肽基因Ahpm-2进行了优化。随后,将优化基因克隆入表达载体pET32a中高亲水酸性标签Trx(pI=5.27)基因的下游,目标多肽AHPM-2以Trx融合蛋白的形式在E.coli Origami (DE3)中成功获得可溶表达。适宜诱导条件为:诱导前OD_(600)值0.6～0.8,0.1 mmol/L IPTG,37℃,诱导5 h。该条件下,表达的Trx融合蛋白占到胞内总蛋白的50%以上,其中90%以上以可溶的形式表达。经镍柱和凝胶柱纯化后,目标蛋白的纯度达到95%以上,并可从1 L发酵液中获得约200 mg高纯Trx-AHPM-2。Trx融合蛋白经胃肠模拟消化后的水解液具有极强的ACE抑制活性(IC_(50)=4.5μg/mL)。UPLC-MALDI-TOF-TOF分析表明AHPM-2中带有Pro残基(处在倒数第三位)且C-末端或N-末端为芳香族氨基酸(Trp、Tyr和Phe)或Leu残基的活性六肽(如IFVPAF和FALPQY)易于胃蛋白酶高效率释放,但是C-末端为脯氨酸(Pro)残基的活性肽段(如IKP)难以从多聚体中释放,并且两条活性肽序列中各自带有的Pro残基之间的距离靠得太近也不利于活性肽序列的完整释放。
3.针对降血压肽多聚体中C-末端为Pro残基的高活性肽段难以释放的问题,研究根据ACE对其底物C-末端二肽的选择性,以IKP为模式三肽,设计并合成了六条ACE抑制五肽IKPVQ、IKPVA、IKPVK、IKPVR、IKPFR和IKPHL,其IC_(50)值均在1.5μmol/L以下。UPLC-MADIL-TOF-TOF质谱分析证实这些五肽均能在ACE的作用下释放IKP,释放率从23%到84.6%。另外,五肽中的Pro-Val和Pro-Phe肽键相对于Pro-His肽键更易于ACE的切割,且底物肽C-末端的碱性氨基酸残基能显著提高切割效率。通过胃肠消化模拟实验后,质谱分析表明这六条五肽均能抗胃蛋白酶的消化,但除了IKPVA(IC_(50)=0.94μmol/L)对胰酶具有80%的抗性外,其它五肽均被胰酶降解,其ACE抑制活性受到不同程度的影响。其中,IKPFR (IC_(50)=0.33μmol/L)在胰酶的作用下产生62.1%的IKPF(IC_(50)=4.6μmol/L)与37.9%的IKP(IC_(50)=0.68μmol/L),仍然具有很强的ACE抑制活性(IC_(50)=2.01μmol/L)。抑制动力学研究显示IKPVA和IKPFR均为竞争性抑制剂,能与ACE的天然底物竞争结合ACE的活性位点。以上结果表明IKPVA和IKPFR可用于构建降血压肽多聚体,并能在ACE或胃肠酶的作用下释放出高降压活性肽IKP。此外,二肽VA和FR有望作为连接片段用以辅助其它C-末端为Pro残基的活性肽段从其前体多肽中高效率释放。
4.为了筛选出既本身抗胃肠酶消化,又能被胃肠酶或ACE从其前体多肽中高效率释放的活性肽单体,研究选择了蛋白序列中具有高比例疏水性氨基酸(43.4%)和碱性氨基酸(13.7%)残基的蛋清溶菌酶进行胃肠消化模拟实验,发现其胃肠酶水解液发挥了很强的ACE抑制活性,IC_(50)值达到了15.6μg/mL。通过超滤、制备型RP-HPLC、分析型RP-HPLC及MALDI-TOF-TOF从溶菌酶的胃肠酶水解液中分离鉴定出三种ACE抑制肽MKR、RGY和VAW,其IC_(50)值分别为25.7﹑61.9和2.86μmol/L,均为首次报道。ACE温育实验结果表明VAW和MKR为真实型的ACE抑制肽,而RGY为底物型ACE抑制肽。抑制动力学研究显示VAW、MKR和RGY均为竞争性抑制剂。以上结果表明该研究鉴定的ACE抑制肽有望被作为设计降血压肽多聚体的活性肽单体,同时也揭示了蛋白原料和酶的组合选择对于筛选适宜目标肽段的重要性。
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 OD_(600) 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 IC_(50) 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 OD_(600) 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 Ni~(2+) 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 IC_(50) 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 IC_(50) 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 (IC_(50)=4.6μmol/L) and IKP (IC_(50)=0.68μmol/L) by pancreatic digestion, and the corresponding hydrolysate still maintained a potent ACE inhibitory activity (IC_(50)=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 IC_(50) value of 15.6μg/mL Three novel ACE inhibitory peptides, MKR, RGY and VAW respectively with IC_(50) 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.
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