Prokaryotic Expression、Purification And Activity Analysis Of Recombinant Antihypertensive Peptide

Hypertension is one of the main factors leading to stroke,chronic renal failure,myocardial infarction and other diseases,which has seriously affected human health.The main drugs available on the market to combat hypertension are nifedipine,captopril,and compound antihypertensive tablets,but because of their serious side effects,such as causing rashes,slow or rapid heartbeat,and dry cough,people are increasingly advocating healthier treatments.AngiotensinⅠ-converting enzyme（ACE）plays a significant role in the regulation of blood pressure and antihypertensive peptides,which are derived from food proteins,can lower blood pressure by inhibiting the activity of ACE enzyme.Because of its specificity,high safety,effectiveness and no side effects,antihypertensive peptides have gradually become a research hotspot.In recent years,scholars have mainly prepared and identified antihypertensive peptides by hydrolyzing food-derived proteins with proteases.Due to the small proportion of antihypertensive peptides in protein,the difficulty of separation and purification and low yield,the industrial preparation of antihypertensive peptides has been limited.The rapid development of genetic engineering technology brings great prospect for the production of antihypertensive peptides.In this study,the antihypertensive peptide WQVLPNAVPAK（Trp-Gln-Val-Leu-Pro-Asn-Ala-Val-Pro-Ala-Lys）was used as the parent peptide.The recombinant plasmid p ET30a-ACEIP was constructed by linking the gene of the recombinant antihypertensive peptide amplified by PCR to the expression vector p ET30a.which was transformed into Escherichia coli BL21（DE3）.The gene engineering expression strain called E.coli BL21（DE3）/p ET30a-ACEIP was induced by IPTG.The induction expression,purification and hypotensive effect of recombinant antihypertensive peptide ACEIP were investigated.The main research and conclusions of the study are as follows:（1）The active peptide WQVLPNAVPAK,which was confirmed to have hypotensive effect by in vitro ACE inhibition assay and in vivo experiments in SHR,was selected and converted into gene sequence by tandem formation of hypotensive peptide muiltimer ACEIP according to the enzyme cleavage site of trypsin.The sequence was optimized according to the codon preference of Escherichia coli and then synthesized artificially.The plasmid p ET30a-ACEIP was constructed by double digestion,purification and ligation of the required fragments.Respectively,a positive recombinant bacterium E.coli BL21（DE3）/p ET30a-ACEIP was obtained after transformation.The recombinant gene was identified by colony PCR and sequencing.The results showed that the target gene expressed by the engineered bacterium was consistent with the designed gene without error code and code shift.（2）The constructed recombinant strain E.coli BL21（DE3）/p ET30a-ACEIP was induced with IPTG for expression and the effects of expression conditions:medium,IPTG induction concentration,temperature and time on the expression of the engineered strain were also investigated.The protein expression was analyzed by 16.5%Tricine-SDS-PAGE electrophoresis.Electrophoresis results showed that compared with the empty plasmid-expressing engineering bacteria and the strains without IPTG-induced expression,the engineered bacteria E.coli BL21（DE3）/p ET30a-ACEIP induced by IPTG showed a clear band at a molecular weight of about 8.7 k Da,which was consistent with the designed target gene and Western Blot further indicated that the recombinant hypotensive peptide ACEIP was successfully expressed in E.coli BL21（DE3）.At the same time,by optimizing the fermentation conditions of the engineered bacteria and orthogonal experimental design,the optimal culture conditions were:LB was used as the medium for subsequent experiments,and the highest expression of recombinant protein was achieved at a concentration of 0.8 mmol/L isopropylβ-D-thiogalactopyranoside at 37°C for 8 h,accounting for 38%of the total protein of the engineering bacteria.（3）The separation and purification process of recombinant peptide ACEIP was studied.Since the peptide ACEIP was expressed in the form of inclusion bodies,it was treated with washing solution I and II,lysis solution,and then purified by Ni²⁺affinity chromatography.The concentration of the purified antihypertensive peptide was determined by BCA method,which showed that 61.30 mg of recombinant peptide could be obtained from 1 L of engineered bacterial fermentation broth.The recombinant peptide solution was subjected to gradient multiplex,dialysis with 1×enterokinase digestion buffer,recombinant enterokinase digestion,and then the histidine and enterokinase tags were removed by adsorption through nickel column affinity chromatography again to obtain pure recombinant tandem protein ACEIP.Its purity was over 90%and its expression was 57.84 mg/L.It was digested with trypsin,and the digested solution was subjected to reversed-phase high-performance liquid chromatography for the content of the monomer was measured as 50.00 mg/m L.（4）The in vitro ACE inhibitory activity of recombinant peptide was studied.The in vitro ACE inhibition rate of the enzymatic peptide was determined by HPLC of the generated hippuric acid content using marnesyl-histidine-leucine as the reaction substrate.The results showed that the IC₅₀ of the enzymatic peptide was 6.39 mg/m L.In vitro simulated digestion experiment showed that the hydrolysate of the recombinant peptide digested by gastrointestinal protease also had a strong ACE inhibition rate of more than 70%.It indicates that the recombinant peptide could release the active hypotensive fragment through the action of gastrointestinal protease.