Prokaryotic Expression And Purification Of Antihypertensive Peptide IYPR And Analysis Of Its Activity

Antihypertensive peptides are the inhibitors of angiotensin I-converting enzyme(ACE).They can exert an antihypertensive effect as the consequence of regulating the rennin angiotensin system(RAS)and bradykinin kinin system(BKS)by inhibiting the activity of ACE.These peptides are welcomed by patients for the advantages of being safer,easily absorbed,stable,and special without the side effects.Up to now,many antihypertensive peptides were obtained by means of enzymatic hydrolysis.To obtain antihypertensive peptides with high purity,the processes for purification are often time-consuming,laborious,and involve lengthy operation techniques.In order to provide foundation for further investigation of new antihypertensive medicine,the aims of this study were:1)to construct a recombinant engineering bacterium for the expression of IYPR(Ile-Tyr-Pro-Arg),2)to study its expression level under different conditions,3)to purify the antihypertensive peptide and analyze its ativity,4)to investigate the effect of ultrasound on the growth and recombinant protein expression of recombinant Escherichia coli(E.coli).The main contents and results of this research are as follows:(1)Based on the amino acid sequences and the codon usage bias of E.coli BL21(DE3),a DNA fragment encoding the peptide IYPR was designed and synthesized.The recombinant vector pET-30a(+)-IR was constructed by reactions of restriction enzyme digestion and ligation.The clones were selected and confirmed by PCR reaction and DNA sequencing after the transformation of pET-30a(+)-IR into E.coli cells.Results showed that the target gene was consistent with the design without error code and frame shift.(2)The expression profiles of the recombinant protein under different IPTG concentrations,different induction time,additives and different compositions of medium were investigated.Tricine-SDS-PAGE analysis was used to analyze the expression level.Compared with that of the non-induced culture,an obvious accumulation of expression protein band was observed at about 8.0 kDa in Tricine-SDS-PAGE analysis,which was in high agreement with the predicted molecular weight.LB was chosen as the optimum medium among the five kinds of media such as LB,TB,SOC,SOB and 2xYT.Addition of 5 mmol/L EDTA?1%glucose?5 mmol/L Ca2+ and 5 mmol/L Mgig in the media did not result in a significant change in the recombinant protein expression.The resulting expression level of the recombinant protein accounted for about 29%of cellular protein at a temperature of 37 ?,IPTG concentration of 0.6 mmol/L and induction time of 9 h.(3)The technology of separation and purification of the antihypertensive peptide IYPR was studied.Inclusion bodies were extracted by centrifugation,ultrasonic crushing and careful washing.The inclusion body protein was purified by Ni2+-NTA affinity chromatography column.The purified recombinant protein was cleaved by enterokinase and the His-tag was removed by dialysis.Target tandem peptide was mixed with trypsin and purified by Sephadex G-15 column.The purified peptide was analyzed by ESI-MS and MS-MS for molecular mass and amino acid sequence determination.The relative molecular mass of the peptide was 548.23 Da and amino acid sequence was Ile-Tyr-Pro-Arg.(4)In order to realize the application potential of recombinant antihypertensive peptide IYPR(Ile-Tyr-Pro-Arg),more information is needed regarding the activity under different conditions including temperature,pH and gastrointestinal proteases encountered in the process and manufacture.In ACE inhibition assays,FAPGG was used as the substrate for the assays of ACE inhibitory activity of IYPR.The information has also been realized regarding the activity of purified IYPR under different conditions including temperature,pH,gastrointestinal proteases and the inhibition pattern.The obtained IC50 value for the peptide was found to be 61 mg/L.When the sample was heated at 90 ? for 1 h,the activity of IYPR was reduced to 98%of the original.Incubation in aqueous solutions(from pH 2 to 10)resulted no significant change in activity of IYPR.When the peptide was digested by pepsin,its activity was increased by 13.0%.Howerer,incubation with trypsin resulted no change in its activity.ACE inhibiton pattern of IYPR was found to be competitive by Lineweaver-burk plot analysis.(5)Antihypertensive activity of different doses of peptide was evaluated by measuring the changes of systolic blood pressure(SBP)in spontaneously hypertensive rats(SHR)after single oral administration and continuous oral administration.The contents of glucose,triglyceride and cholesterol in serum and the hematologic indices of SHR were also investigated.The administration of 400 ?g/kg and 1000 ?g/kg of peptide IYPR resulted in a significant decrease of the SBP in SHR(P<0.05),and the activity was maintained for more than 8 h.A reduction of 50 mm Hg of SBP was observed at 4 h after single oral administration of 400 ?g/kg peptide(P<0.01).SHR and SD rats were daily given 400 ?g/kg of IYPR for 35 days.The blood pressure was measured weekly during the study period by the tail-cuff method.IYPR lowered SBP in SHR rats during the investigation period,and there was no significant change of SBP in SD rats.A reduction of 44 mm Hg of SBP was observed after continuous oral administration of 400 ?g/kg peptide for three weeks.This effect was better than that caused by 200 mg/kg peptide from porphyra yezoensis and similar to that caused by 30 mg/kg of captopril,and the doses of porphyra yezoensis and captopril were 500 times and 75 times of IYPR.After continuous oral administration,no changes were observed(P>0.05)in the contents of glucose,triglyceride and cholesterol and the hematologic indices of SHR and SD rats by comparing with the control group.Paraffin sections of heart,liver and kidney were observed by hematoxylin-eosin staining(HE)staining and microscope.The results showed that there were no changes in the control group and treated group.(6)In this study,it was aimed to investigate the effect of ultrasonic stimulation on the growth and protein expression of recombinant E.coli in suspension culture.The growth was measured by spectrophotometric method and the protein expression was dectected by Tricine-SDS-PAGE analysis.Results show that the growth of E.coli was more promoted by sweeping ultrasound than by fixed ultrasound.The cells were stimulated by ultrasound at 28±2 kHz for 30 min at period 1,under which the growth was obviously promoted by ultrasonic stimulation over the control.The protein expression was not increased by the ultrasound agitation for 5,30,and 60 min at period 1.When the ultrasonic stimulation occurred at the time of optical density at 1.00(period 2),the cell growth and protein expression were inhibited.However,when the cells were inducted by IPTG for 2 h(period 3)and stimulated for 5,30 and 60 min,ultrasound resulted in no changes in the cell growth and protein expression.Fura-2/AM fluorescence probe was used to determine the change in intracellular Ca2+concentration.Research result showed that concentration of Ca2+ was decreased to the lowest point by ultrasound treatment for 30 min.The data obtained show that it is reasonable for the tandem expression of peptide IYPR in E.coli.The purified peptide can exert good activity and decrease the SBP in SHR.All these provide a theoretical basis for further development of peptide IYPR into an effective antihypertensive agent for prevention and treatment of hypertension.